Thermodynamics of Protein

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
Vol. 24, Nos. 1 & 2, pp. 152, 2004
REVIEW
Thermodynamics of ProteinLigand Interactions:

History, Presence, and Future Aspects
Downloaded By: [University of Missouri] At: 19:42 13 May 2009
Remo Perozzo,* Gerd Folkers, and Leonardo Scapozza
Department of Chemistry and Applied BioSciences, Swiss Federal

Institute of Technology (ETH), Zurich, Switzerland
ABSTRACT
The understanding of molecular recognition processes of small ligands and

biological macromolecules requires a complete characterization of the binding
energetics and correlation of thermodynamic data with interacting structures
involved. A quantitative description of the forces that govern molecular
associations requires determination of changes of all thermodynamic parameters,
including free energy of binding (G), enthalpy (H ), and entropy (S ) of
binding and the heat capacity change (Cp). A close insight into the binding
process is of significant and practical interest, since it provides the fundamental
know-how for development of structure-based molecular design strategies. The
only direct method to measure the heat change during complex formation at
constant temperature is provided by isothermal titration calorimetry (ITC). With
this method one binding partner is titrated into a solution containing the
interaction partner, thereby generating or absorbing heat. This heat is the direct
observable that can be quantified by the calorimeter. The use of ITC has been
limited due to the lack of sensitivity, but recent developments in instrument design
permit to measure heat effects generated by nanomol (typically 10100) amounts
of reactants. ITC has emerged as the primary tool for characterizing interactions
*Correspondence: Remo Perozzo, Department of Chemistry and Applied BioSciences, Swiss

Federal Institute of Technology (ETH) Zurich, Winterthurerstr. 190, CH-8057 Zurich,
Switzerland; Fax: 41-1-6356884; E-mail: remo.perozzo@pharma.ethz.ch.
DOI: 10.1081/RRS-120037896 1079-9893 (Print); 1532-4281 (Online)

Copyright & 2004 by Marcel Dekker, Inc. www.dekker.com
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2 Perozzo, Folkers, and Scapozza
in terms of thermodynamic parameters. Because heat changes occur in almost all

chemical and biochemical processes, ITC can be used for numerous applications,
e.g., binding studies of antibodyantigen, proteinpeptide, proteinprotein,
enzymeinhibitor or enzymesubstrate, carbohydrateprotein, DNAprotein
(and many more) interactions as well as enzyme kinetics. Under appropriate
conditions data analysis from a single experiment yields H, KB, the
stoichiometry (n), G and S of binding. Moreover, ITC experiments performed
at different temperatures yield the heat capacity change (Cp). The informational
content of thermodynamic data is large, and it has been shown that it plays an
important role in the elucidation of binding mechanisms and, through the link to
structural data, also in rational drug design. In this review we will present a
comprehensive overview to ITC by giving some historical background to
calorimetry, outline some critical experimental and data analysis aspects, discuss
the latest developments, and give three recent examples of studies published with
respect to macromoleculeligand interactions that have utilized ITC technology.
Key Words: Isothermal titration calorimetry; Proteinligand interaction;

Thermodynamics.
INTRODUCTION
A fundamental principle of all biological processes is molecular organization

and recognition. Biological macromolecules are able to interact with various small
and large molecules, with a high degree of specificity and with high affinity,
fascinating chemists and biologists from the very beginning of modern biochemistry.
A prerequisite for a deeper understanding of the molecular basis of proteinligand
interactions is a thorough characterization and quantification of the energetics
governing complex formation. Calorimetry is the only technique enabling us to study
directly the basic physical forces between and within a macromolecule in sufficient
detail by measuring heat quantities or heat effects.
Historical Background to Thermodynamics and Calorimetry
The background of the development of calorimetry and thermodynamics has

been the subject of a variety of historical studies, and here we try to do a short
summary of the most interesting aspects thereof (19). Calorimetry is a very old
science. In principle, the historical development of calorimetry and thermo-
dynamics began with the description and definition of temperature and heat. The
first known documents from the early 17th century witness for very crude attempts
to describe temperature, most of them derived by perception: heat of a breeding
hen, heat of boiling water, heat of glowing charcoal. These estimations were too
rough, and therefore it was necessary to develop objective standards. The invention
of the first thermometer had its origin in the same time period. The concept of
expansion of gases and liquids due to heat was already known from the antiquity
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and was used by Galileo and Drebbel. They independently used a bulb with an
open-ended stem inverted over water to observe the expansion of air. The results
obtained with these so-called thermoscops were very inaccurate, confused with the
effects of barometric pressure and lacking scaling. The next crucial step to make
satisfactory thermometers was the use of (pure) liquids instead of air (water,
ethanol, mercury) in a closed compartment. By the end of the 17th century, a
reliable temperature scale was established by Fahrenheit and Celsius. It was
around this time when the nature of heat and its quantitative aspects became of
interest.
People had speculated on the nature of heat since ancient times. It was a
widespread belief that heat was a substance, some held the view that it was composed
of atoms. During the 18th century the foundations of calorimetry were laid by
Joseph Black. He preferred an alternative explanation of heat being a fluid that can
be absorbed or squeezed out of bodies and can flow from one place to another. He
recognized that heat applied to melting ice did not change the temperature of the
mixture but was consumed for the solidliquid phase transition, for the first time
clearly discriminating between the strength and amount of heat. Black
introduced the concept of latent heat and showed that quantities of heat could be
estimated from the amount of melted ice. This view brought him to the first
calorimetric experiments with a simple phase-transition calorimeter. A warm probe
was placed in the cavity of an block of ice, covered with a plate of ice and brought to
thermal equilibrium. Furthermore, he adopted the idea of mixing water of different
temperatures (mixing calorimeter) from Brooke Tylor (1723) to determine a series of
latent heats of different substances.
At the same time, A. L. Lavoisier and P. S. Laplace became interested in the
theory of heat. They considered the widespread mixing calorimeters as unsuitable
because of several disadvantages: the need of delicate corrections for the heat
capacity of vessel and thermometer, heat loss by cooling, chemically reacting
substances, inmiscible liquids. Moreover, this method did not allow the
measurement of the heat produced during combustion and other chemical
reactions, and during respiration, topics in which they were mostly interested.
They developed the first convenient phase transition calorimeter that led to
reproducing results. It was a simple but ingenious ice calorimeter, a device for
measuring heat release due to respiration and combustion (Fig. 1). The instrument
consisted of a chamber surrounded by an ice-packed jacket, and the whole device
was further insulated with another ice-packed jacket to improve accuracy. The
amount of water collected from the melted ice of the inner jacket was used as a
measure of the heat evolved in the chamber. The handling was difficult and
experiments could only be performed on days when the outside temperature was a
few degrees above freezing. With this device, Lavoisier and Laplace determined the
specific heat of various substances and found fairly good results compared to
modern standards. The most famous experiments were conducted around 1780,
when Lavoisier and Laplace measured the heat generated by a guinea pig and
determined the amount of carbon dioxide in its exhaled air during the experiment.
They compared it to heat release and carbon dioxide formation when burning
charcoal. The results were accurate enough to conclude that respiration was a form
of combustion.
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Figure 1. The ice calorimeter of Lavoisier and Laplace (from Oeuvres de Lavoisier, Tome
Premier, Paris, Imprimerie Imperiale, 1862).
Despite this interesting experimental work, the resulting interpretations of the

nature of heat remained unclear. Lavoisier still treated heat as a weightless substance
and called it caloric, matter of fire. Laplace favored a mechanical explanation of
heat as motion of particles of matter, a view that emerged toward the end of the
18th century out of experimental evidence provided by Count Rumford, formerly
known as Benjamin Thompson. Rumford noticed that drilling a hole into metal to
build a cannon barrel generated a large quantity of heat, and he noticed that there
was no limit to the amount of heat that could be produced by simple drilling. He
concluded that heat was motion and not matter (or caloric), otherwise it had to stop
when the cannon was running out of caloric. But there was a big controversy about
this theory, and it was not until the middle of the 18th century when the caloric
theory was finally overthrown. The kinetic gas theory was established and the
concept of energy arose.
With the Industrial Revolution beginning in the 19th century, the nature of
matter became of more than academic interest. With the realization that heat from
combustion could produce work, the science of thermodynamics was born. It is
concerned with the rules governing the interconversion of energy and is able to
predict the feasibility of chemical processes.
Calorimetric measuring techniques remained more or less the same during
this time period, although there were some modifications and improvements. In
the last few decades, calorimetric techniques have started to become of interest to
biochemists and biologists outside a few specialized laboratories. Since practically
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every process, be it physical, chemical, or biological, is accompanied by heat

changes, it is obvious that calorimetry could serve as powerful analytical tool for a
variety of applications, particularly in biological sciences. With concurrent advances
in molecular biology, expression and purification techniques, that made available
significant amounts of homogeneous protein, there was an increasing need for more
and reliable thermodynamic data. This gave the inputs to develop new and very
sensitive calorimeters, requiring only small sample quantities and being able to detect
accurately very small heat quantities.
Since the middle of the 20th century several calorimetric principles of different
practical design have emerged. But it is only since the last few years, with the
development and improvement of sufficiently sensitive, stable, user friendly, and
affordable commercial calorimeters, that made calorimetry to become an almost

routine analytical procedure in biochemical and biophysical research. Since modern
instruments are very sensitive, detecting heat changes in the range of microcalories,
requiring only 10100 nmol of sample in a volume of 0.21.4 mL, they are usually
denominated as microcalorimeters.
CALORIMETRIC PRINCIPLES AND PROPERTIES
To avoid confusion in describing the principles of calorimetry and calorimeters

it is useful to distinguish three important areas: the measuring principle, the
operating mode, and the type of construction (5,1012).
Principles of Measurement
Calorimeters are instruments for quantification of heat effects. Several principles

of measurement have come into use. Amongst solution calorimeters there are two
main groups: adiabatic calorimeters and heat conduction calorimeters. With an ideal
adiabatic calorimeter there is no heat exchange between the calorimeter and the
surroundings, and the heat quantity Q evolved during the experiment is directly
proportional to the observed temperature change T, and to the heat capacity " of
the reaction vessel and its contents:
Q " T 1
Thus, in an experiment the heat quantity is determined by measuring the
temperature change. In an ideal heat conduction calorimeter the heat evolved is
quantitatively transferred from the reaction vessel to the heat sink, a body
surrounding the calorimeter which is usually made of metal. With this type of
calorimeter, some property proportional to the heat flow between vessel and heat
sink is measured. Normally the heat flow is recorded by placing a thermopile wall
between the vessel and the surrounding sink. The temperature difference over the
thermopile gives rise to a potential or voltage signal S that is proportional to the heat
flow. The time integral for the heat flow, multiplied by a calibration constant ",
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is proportional to the heat quantity released in the experiment:

Z
Q " S dt 2
The heat quantity is thus proportional to the area under the signal time curve.
Operating Mode and Construction Design of the Instrument
The most common type of calorimeter in use is the isoperibol calorimeter, also
called constant temperature environment calorimeter. The vessel is separated by
thermal insulation from the surrounding thermostated bath, which forms

the isothermal jacket. The insulation is usually filled with air or vacuum.
Exothermic or endothermic processes will result in a temperature change that is
recorded by a thermometer. In practice, there will always be a small heat loss from
the vessel into the surrounding. Therefore, this calorimeters are not truly adiabatic,
but quasi-adiabatic. The heat exchange cannot be neglected and must be corrected
for. Isoperibol calorimeters are very simple and for fast processes also very precise
instruments. They are used as reaction or solution calorimeters and as combustion
calorimeters, but have not found widespread use in biochemical or biological studies.
In an adiabatic shield calorimeters the reaction vessel is enclosed by an additional
thin-walled metal envelope, the adiabatic shield. It is placed in the vacuum or air
space between the reaction vessel and the thermostated bath. The temperature
difference between the shield and the vessel is kept at zero during the experiment by
automatically applying a suitable heat effect on the shield.
Calorimeters can be in a single or a twin arrangement. Although the single
arrangement is simpler, the twin calorimeter has some advantage which makes it
very attractive for microcalorimetry. One of the calorimetric vessels, the reaction
cell, contains the system of interest, whereas the other vessel, the reference cell,
contains water or buffer. With such an arrangement the recorded signal is a
differential signal, of which the effects of thermal disturbances from the
surroundings are expected to cancel out.
ISOTHERMAL TITRATION CALORIMETRY
The main calorimetric techniques applied to investigate biological macromole-

cules are differential scanning calorimetry (DSC) and isothermal titration
calorimetry (ITC). DSC measures the enthalpy and heat capacity of thermal
denaturation, and researches have learned about stability of biological macro-
molecule (proteins and nucleic acids) and of macromolecular assemblies (1316).
In contrast, ITC measures the heat evolved during molecular association. The
direct thermodynamic observable is the heat associated with a binding event, i.e., a
ligand is titrated into a solution containing the macromolecule of interest and
the heat evolved or absorbed is detected. It allows the simultaneous determination of
the equilibrium binding constant (KB) and thus the standard Gibbs free energy
change (G), the enthalpy change (H ), the entropy change (S ), as well as the
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stoichiometry (n) of the association event. Moreover, experiments performed at

different temperatures yield the heat capacity change (Cp) of the binding reaction
(1719). As almost any interacting system is characterized by changes in enthalpy,
there is a vast range of potential ITC applications.
ITC Instrumentation
A number of suppliers offer microcalorimetric instruments with sufficient

sensitivity for the determination of binding reactions, but most studies published so
far have used instruments from MicroCal (Northhampton, MA, USA). With the
introduction of the commercially available Omega titration calorimeter in 1989 (19),

titration calorimetry has had a broad impact throughout biotechnology, which is
reflected by a large body of publications. The Omega unit has been subsequently
modified and automated during the last decade, offering now the most sensitive
instrument that is able to measure interaction heat effects of reactant concentrations
as low as 110 nmol. This type of calorimeter is based on a cell feedback that
measures the differential heat effects between a reference and sample cell. A constant
power applied to the reference cell activates the power feedback circuit that in turn
regulates the temperature in the sample cell, thus slowly increasing the temperature
during a measurement (typically less than 0.1 C/h). The resting power applied to the
sample cell is the baseline signal.
Exothermic reactions as a result of the addition of ligand will decrease the
necessary feedback power, and endothermic reactions lead to an increase in feedback
power. The enthalpy of reaction for each injection is obtained by integration of the
deflections from the resting baseline. Both cells are accessible by long narrow access
tubes through which samples are introduced or removed using long-needled syringes.
Typically, the reference cell is filled with water and the sample cell with the system of
interest. The ligand is applied by injection syringes with long needles having a
stirring paddle attached to the extreme end. The syringe is continuously rotated
during an experiment, leading to complete mixing in the cell within a few seconds
after an injection. The mechanical heat of stirring is constant and becomes part of
the resting baseline. With optimal performance (short equilibration time) a complete
binding isotherm may be determined within 30 min, although in practice it takes
usually 80100 min to obtain reliable data.
It is worth emphasizing that calorimetric binding experiments are very challenging
since noncovalent binding heats are intrinsically small, typically in the range of
510 kcal mol1, and must be liberated stepwise during the binding experiment.
Furthermore, ligand addition produces additional heat effects arising from dilution and
mixing, for which corrections must be made, and which are frequently comparable to
the binding heat of interest. Considering the case of a typical reaction of interest
that exhibits the heat effect of 5 kcal mol1 in a 12-ml solution containing 107 mol
of protein (a few mg), the experiment would liberate about 0.5 mcal of heat after
complete saturation of all binding sites. Assuming that this heat is released
upon 10 injections, the mean individual contribution would be 50 mcal. Accurate
detection, with an accuracy of 10% or better, of such small quantities would require
instrumental sensitivity and noise levels as low as 5 mcal or less. This corresponds to
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temperature changes in the sample solution of just a few millionths of a degree and
is comparable to the inevitable heat effects of dilution, mixing, and stirring. The
absolute detection limit of the latest generation VP-ITC calorimeter, expressed as
the minimum detectable heat quantity, is reported to be 0.1 mcal (20).
EXPERIMENTAL DESIGN
General Experimental Setup
The setup of an ITC experiment is largely dependent on the thermodynamic

characteristics of the system of interest, i.e., the expected binding affinity and the
heat effect of the interaction. The appropriate concentration range for the
macromolecule placed in the cell depends on the binding constant of the reaction.
The shape of the binding curve is dependent on the product of the binding constant
KB (in M1) and the molar concentration of macromolecule [MT] being titrated (19):
C KB MT 3
The sensitivity of the shape of the binding isotherm to the dimensionless
parameter C is crucial for determination of the binding constant. At high values for
the so-called C-value (C > 500), the shape of the curve approaches a step function
and becomes increasingly insensitive to changes in KB (Fig. 2). Experience shows
that conditions should be chosen to have a C-value in the range of 10100 for an
accurate determination of KB. Therefore, to measure at these C-values, very strong
1
0
-1
-2
kcal/mol of injectant
-3
-4 C=1
-5
-6 C = KB[MT]
-7
-8
C=10 C=50
-9 C=1000
C=500
-10
-11
0 1 2
Molar Ratio
Figure 2. Simulated calorimetric binding curves illustrating the dependence of the shape of
the curve on the product of the association constant KB and the total macromolecule
concentration MT (C KB [MT]). The curves are simulated for several C-values (as indicated
in the plot) according to Eqs. (16) and (18) for H 10 kcal mol1. For high C-values the
binding isotherms approach a step function, becoming increasingly insensitive to changes
in KB. At low C-values, the binding curve becomes a horizontal trace that yields very little
information about KB, making it necessary to use high macromolecule concentrations to
obtain suitable binding isotherms.
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binding (107108 M1) requires low concentrations of the macromolecule. With

decreasing concentrations of the reactants, the signal arising from the interactions
will also become smaller, leading to the detection limit of ITC. This gives the
technical limit of the highest affinity constant that can be determined.
At low C-values (C < 10), the binding curve forms a horizontal trace that again
yields very little information about KB. Consequently, it is necessary to use high
macromolecule concentrations to obtain informative binding isotherms if low binding
affinity is expected. In principle, there is no lower limit to lower binding affinity events
that can be determined, but in practice there are problems with solubility, stability,
and often with availability of the macromolecule in order to measure in the C-value
range of 10100. From Fig. 2 it is evident that even at low ligand concentration, only a
minor fraction of ligand is bound to the macromolecule, making it difficult to detect

sufficient heat and to determine H accurately. This often sets the limit of the lowest
affinity constant measurable in the range of 104 M1.
The correct choice of reactant concentrations depends not only on the
magnitude of KB, but also on the objective of the experiment. If it is of interest to
simultaneously determine KB, H, and n for a binding event, a complete binding
isotherm must be recorded. Considering the limiting sensitivity of 0.1 mcal, each
injection should produce an average heat change of 12 mcal in the 1.4-ml cell. For
a series of 10 injections, each of 10 mL, a total Q of 20 mcal in the sample volume
are required to define a total binding curve:
Q HMT V0 4
where H is the enthalpy of binding and V0 is the reaction volume of the sample cell.
Solving Eq. (4) for [MT] predicts a minimum concentration of about 1.4 mM for
a protein with a H of 10 kcal mol1 needed to generate a complete binding
isotherm to yield n, KB, and H. According to Eq. (3), an arbitrarily chosen KB of
106 M1 would result in a very low C-value of 1.4. In practice it will be necessary to
increase the protein concentration to perform the experiment in the ideal range for
C-values of 10100. As a desirable side effect the heat signals will become larger. If
the same calculation were done using higher affinity (107 M1), the resulting C-value
of 14 would be sufficient for a complete deconvolution of the binding isotherm.
Taken together, the almost 10-fold increase in sensitivity achieved from first to third
generation microcalorimeters allows now to measure higher affinity (up to 109 M1)
faster, more accurately, and with less material.
For some applications it is preferred to obtain H not as a fitting parameter,
but to directly determine H very accurately. In this case it is common practice to
measure H at concentrations when the binding partners are fully associated and
the saturation is still low, i.e., full association at partial saturation (21). At these
conditions (C-value >100), the amount of heat released or absorbed is directly
determined by the amount of ligand injected:
Q HLT Vinj 5
where [LT] is the concentration of the ligand solution in the syringe, and Vinj is the
injection volume. In principle, this allows the determination of H with a single
injection. In practice H will be determined as the mean of a number of injections
from the same experiment.
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To obtain high quality data, an appropriate protocol has to be established by

optimizing ligand and protein concentrations and the injection volume. Typically, the
ligand concentration is much higher since several equivalents must be added to the
sample cell. The titration experiment should be planned to approach or reach
complete saturation of the binding sites at the end of the experiment. To generate a
sufficient number of data points, which will improve data analysis, the ligand has to be
added in small aliquots. However, the heat signal should not become too small to
maintain high precision of each data point. If the interaction heat is small, it will be
necessary to choose larger injection volumes. These prerequisites define the titration
protocol, and it is up to the experimentator to find the ideal compromise. As a rule of
thumb, 25 injections, each of 5 mL, of a ligand solution with a concentration 25 times
higher than that of the protein solution will result in an adequate binding isotherm. If
the ligand is poorly soluble, it is possible to place it in the sample cell and to inject the
macromolecule. As long as the binding stoichiometry is 1:1, either interacting
molecule can act as the titrant without adjusting the binding model. For more
complicated cases where this assumption does not hold, the model must be modified
accordingly (22).
The time between successive injections is another important parameter. If
association is rapid, the instrument baseline will be equilibrated in a short time,
depending on the response time of the calorimeter. Under such conditions 34 min
are sufficient to reach baseline again after injection. In contrast, heat signals of slow
processes require much more time to reach thermal equilibrium. Several other issues
related to experimental design should be mentioned. It is crucial that solutions of
ligand and macromolecule are pure and exactly match with respect to pH, buffer
capacity, and salt concentration. This means that macromolecule and ligand are
preferably dissolved in the same buffer. To achieve this goal, it is good practice to
dialyze the protein prior to the experiment and dissolve the ligand in the dialysis
buffer. This procedure will prevent spurious heat effects resulting from mixing of
different buffers. Both interacting components, often purified from biological
source, must be free of contaminating enzymatic activity that could affect the
association event under investigation. Furthermore, the formation of air bubbles has
to be avoided. Thus it is very important to thoroughly degas all solutions prior to the
experiment. Any air in the syringe can cause variation in the injected volume or lead
to additional heat signals, and bubbles in the sample cell interfere with the thermal
contact of solution and cell wall. Finally, in most experiments the heat effect of the
first injection of a series of injections is obviously too small. This results from
diffusion while equilibrating the system. Even if care is taken to avoid this leakage,
the problem may persist. Therefore it is common practice to make a small first
injection of 1 mL and then to remove the first data point before data analysis.
Control Experiments
Isothermal titration calorimetry not only measures the heat released or absorbed
during binding reactions, but it detects the total heat effect in the calorimetric cell
upon addition of ligand. Thus, the experimental heat effect contains contributions
arising from nonspecific effects, such as dilution of ligand in the buffer, dilution of
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11
the protein sample, heat of mixing, temperature differences between the cell and the
syringe, and mixing of buffers of slightly different composition. These contributions
need to be determined by performing control experiments in order to extract the heat
of complex formation. This would need at least a further three titrations to measure
these effects (ligand into buffer solution, buffer into protein solution, buffer into
buffer solution). In practice however, the latter contributions are found to be small
and frequently negligible, whereas the heat of ligand dilution may be significant and
needs to be corrected for. Alternatively, if the titration experiment is designed to
ensure complete saturation of the enzyme before the final injection, and if the blank
experiments mentioned above show the heat of ligand dilution to be concentration-
independent, then the nonspecific heat effects can be estimated very well by
averaging the small heats at the end of the titration.
Evaluation of Protonation Effects
Whenever binding is coupled to changes in the protonation state of the system,

the measured heat signal will contain the heat effect due to ionization of buffer. If the
binding event changes the protonation state of free or bound ligand as well as of free
or complexed macromolecule, proton transfer with the buffered medium occurs. As
a consequence, the heat of protonation/deprotonation will contribute to the overall
heat of binding and Hobs will depend on the ionization enthalpy of the buffer
(Hion). Repeating the calorimetric experiment at the same pH in buffers of different
Hion allows to determine the number of protons nH that are released (nH > 0) or
taken up (nH < 0) by the buffer, and thus to calculate the intrinsic binding enthalpy,
Hbind, corrected for protonation heats:
Hobs Hbind nH Hion 6
In practice, it is recommended to perform ITC experiments in a series of buffers
of different ionization heats under otherwise the same conditions. Values of Hion
have been described (23,24) or can be determined by ITC (25). If the same Hobs is
observed, there is no protonation event coupled to binding. Deviations of Hobs
with different buffers point to a protonation event, and the intrinsic enthalpy of
binding (Hbind) can be obtained from the intercept (Hion 0) of the regression
line described by Eq. (6) (26).
Although it would seem that such buffer effects would mean a nuisance, it is one
of the most powerful means to investigate binding mechanisms, and this property
can be exploited to increase the signal strength of an otherwise undetectable binding
event by simply changing the buffer system to a different pH and to buffers with high
ionization enthalpies (25).
DATA ANALYSIS
The signal monitored by ITC is the differential power applied to the sample cell.
The total heat released or absorbed upon an injection of ligand into the cell
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Time (min)
-10 0 10 20 30 40 50 60 70 80 90 100
0
A
cal/sec -2
-4
-6
-8
kcal/mole of injectant
0
-2
B
-4 Ribonuclease A
-6 Single site binding model
-8 KB(105M1) 2.51 0.029
-10 H (kcal/mol) 12.98 0.02
-12 n 0.953 0.001
-14
0.0 0.5 1.0 1.5 2.0 2.5
Molar Ratio [2CMP]/[RNASE A]
Figure 3. Calorimetric data for the exothermic binding of cytidine 20 -monophosphate
(20 CMP) to ribonuclease A (RNase A) at pH 5.5 (0.2 M K-acetate, 0.2 M KCl) and 28 C
(figure kindly provided by Dr. I. Jelesarov). A: raw data obtained for 25 automatic injections
of 5 mL. Concentrations of RNase A and 20 CMP are 0.145 mM and 3.72 mM respectively. The
area of each peak represents the total heat evolved upon addition of a single aliquot of 20 CMP.
B: titration plot derived from the integrated heats of binding, corrected for heats of dilution.
The solid line represents the nonlinear best fit to the data assuming a single-site binding model.
corresponds to the area under the signal vs. time curve (Fig. 3, panel A). The
instrumental baseline and other unspecific heat effects must be carefully subtracted
from the raw data. Experimental data can be presented as a sigmoid plot (differential
mode) or as a hyperbolic saturation curve (integral mode). The differential
mode treats each injection as an independent point and is plotted as heat evolved
per injection vs. total ligand concentration or the ratio of the total ligand
concentration to the concentration of macromolecule (Fig. 3, panel B). In the
integral mode, the total cumulative heat is plotted against the total ligand
concentration. Fitting a binding model to the calorimetric data plotted in either
mode yields equivalent results. Generally, random errors tend to cancel out in the
integral mode, whereas systematic errors tend to be amplified. Comparative
statistical analysis of both modes can give information about the accumulation of
systematic errors (27,28).
Ligand Binding in Titration Calorimetry
There are many techniques available to measure binding constants (KB).

Equilibrium dialysis, radio-ligand binding assays or ultracentrifugation directly
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13
yield concentrations for the macromolecule [M], ligand [L], or complex [ML]
to calculate KB. Spectroscopic methods are more indirect by detecting an observable
p, the change of which is proportional to the degree of saturation (18,2830).
ITC is the most direct method to measure the heat change on complex formation.
In general, a simple reversible association between a macromolecule M and a
ligand L,
M L $ ML 7
is characterized by its binding constant KB:
ML
KB 8
ML
The observable response of an ITC experiment is the heat change associated

with each addition of ligand. For each injection, the heat released or absorbed
is directly proportional to the total amount of formed complex. This can be
expressed by
q V0 H ML 9
where q is the heat associated with the change in complex concentration, [ML], H
is the molar enthalpy of binding, and V0 is the reaction volume of the sample cell.
In a calorimetric experiment, each addition of ligand gives rise to a heat change
depending on the reaction volume, concentrations, molar enthalpy, binding constant,
heat of dilution, stoichiometry, and the amount of previously added ligand. As the
concentration of unoccupied binding sites begins to decrease, the heat changes
decrease correspondingly as ligand is added. The total cumulative heat after the ith
addition, Q, will be
X
Q V0 H MLi V0 HMLi 10
where [ML]i is the total concentration of complex after the ith injection.
Evaluation of microcalorimetric data requires the consideration of the
observable response in terms of total ligand added or the total ligand concentration.
Therefore, the binding equations must be expressed as a function of total ligand and
macromolecule concentration:
MT ML M 11
LT ML L 12
where [MT] and [LT] are total macromolecule and ligand concentrations,
respectively, and [M] and [L] are free concentrations of macromolecule and ligand,
respectively. [ML] is the concentration of the formed complex.
Single Set of Independent Sites Model
In the simplest case of ligand binding, each macromolecule consists of only one
type of binding sites with a finite number of identical noninteracting binding sites,
ORDER REPRINTS
all of which exhibiting the same intrinsic affinity for the ligand. For such a system,
the binding constant KB is given by

KB 13
1 L
where is the fractional saturation and [L] is the concentration of free ligand. It is
related to the total ligand [LT] and macromolecule concentration [MT], by mass
conservation:
L LT nMT 14
Combining Eqs. (13) and (14) gives the quadratic equation

2 1 LT LT
1 0 15
nKB MT nMT nMT
whose only meaningful root is

0 s 1

1 1 LT 1 LT 2 4LT A
@1 1 16
2 nKB MT nMT nKB MT nMT nMT
The integral heat of reaction Q after the ith injection is given by
Q nMT V0 Hi 17
where V0 is the cell volume and H is the molar heat of ligand binding. The
differential heat of the ith injection is
qi nMT V0 H i i1 18
A nonlinear fit based on Eq. (17) to the hyperbolic saturation curve in the
integral mode (Q vs. [LT]) yields the parameters KB, H, and n from a single
experiment. Based on Eq. (18), the titration data can be fitted to the sigmoid
saturation curve in the differential heat mode (qi vs. [LT], or vs. [LT]/[MT]). The same
parameters are obtained.
Complex Binding Models
Similar relationships as described above exist for other models, i.e., a model for
multiple sets of independent binding sites, single set of interacting sites (cooperative
sites), multiple sets of interacting binding sites. By use of statistical thermodynamic
treatment it is possible to deconvolute a binding isotherm of such complex systems
(3133). Instructive examples from the literature demonstrate the strength of this
approach (3438). However, the success strongly depends on the quality and
reliability of the experimental data.
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15
BASIC THERMODYNAMIC RELATIONSHIPS
The binding enthalpy of proteinligand interactions can be determined

accurately by means of ITC. The association constant KB is related to the Gibbs
free energy G by the well-known relation
G RT lnKB 19
where R is the universal gas constant and equals to 1.987 cal K1 mol1 and T is the
temperature in degrees Kelvin. G is again composed of an enthalpy term (H) and
an entropy term (S), related by another fundamental equation:
G H TS 20
The Gibbs free energy is temperature dependent and is described by

ZT ZT
GT HT0 Cp dT TST0 Cp dlnT 21
T0 T0
where Cp is the heat capacity change and T0 is an appropriate reference

temperature. With Cp being independent of temperature in the range of interest,
Eq. (21) simplifies to

T
GT HT0 TST0 Cp T T0 T ln 22
T0
Equation (22) shows that enthalpy and entropy changes are dependent on
temperature through the heat capacity change Cp:
HT HT0 CpT T0 23
ST ST0 Cp lnT=T0 24

In a thermodynamic analysis the goal is to determine G, H, S, and their
temperature dependence by Cp, since these four parameters provide a full
description of the energetics governing molecular interactions.
STRATEGIES FOR MEASURING

TIGHT BINDING AFFINITY
High-affinity binding constants for proteinligand interactions are inherently

difficult to measure. With increasing affinity, it becomes necessary to work at low
concentration of macromolecule, leading to difficulties in detecting the signal specific
to the analytical method. In the case of ITC, the largest binding constant that can
be measured reliably, approaches 109 M1 for a typical macromoleculeligand
interaction (18,19,39).
The thermodynamic approach offers a powerful advantage for measuring tight
binding affinities and thus Gibbs free energy changes (G). Free energy changes are
state functions, i.e., their values are defined by the initial and final thermodynamic
states, regardless of the pathway connecting the two states. This being the case, it is
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possible to determine the binding constant for a proteinligand interaction under

different conditions that allow measuring the affinity. The result can be corrected to
other conditions of more physiological relevance, if it is known by what parameters
the binding free energy is linked with the conditions being varied. In principal, any
change of physical or chemical conditions that influence ligand association to a
macromolecule is useful, but in most cases linkage of binding with pH and
temperature are exploited.
Linked Protonation Effects in Ligand Binding

Often molecular interactions are very tight (>109 M1), and are not accurately
measurable even with the most sensitive calorimeters available. Generally, molecular
interactions occur to some degree in dependence of pH, reflecting the linkage
between the association of a ligand and the binding of protons (proton linkage). The
molecular basis of the linkage is the result of alterations of pKa values of ionizable
amino acid groups concomitant with binding.
If ligand binding is coupled with uptake of a single proton (Fig. 4), the observed
ligand binding constant Kobs is given as
1 KPc 10pH
Kobs Kint 25
1 KPf 10pH
where Kint is the intrinsic binding constant, KPc and KPf are the proton binding
constants for the complex and free form of the protein and are equal to 10 pKa,c and
10 pKa, f, respectively, of the ionizing group (40,41). According to Eq. (25) proton
linkage can be viewed as change in proton affinity, thus protons will either be
released or absorbed due to ligand binding.
If proton transfer occurs during binding, the Hobs is determined by the
ionization enthalpy of the buffer and the enthalpy of binding corrected for
buffer effects see according to Eq. (6), and both the number of protons (nH) and
Kint
M M:L
Kpf Kpc
M+ M:L+
Figure 4. Scheme for proton binding linked to binding of a ligand L to a macromolecule M.

Ligand binding reactions are shown in horizontal direction whereas proton binding occurs
in vertical direction (see text for details).
ORDER REPRINTS
17
the intrinsic binding enthalpy (Hbind) will vary as a function of pH. Thus nH is
given by
nH f c f f 26
c f
where f and f are the fractional saturation of protons at a given pH of the bound
and free protein. In case of a single protonation event, f c and f f can be expressed as
KPc 10pH
fc 27
1 KPc 10pH
and
KPf 10pH
ff 28
1 KPf 10pH
The change in the number of protons bound by the protein upon binding of the
ligand is the difference between Eqs. (27) and (28):
KPc 10pH KPf 10pH
nH f c f f 29
1 KPc 10pH 1 KPf 10pH
Equation (29) clearly shows that at a minimum of two pH values pKa,c of the
complexed (KPc 10pKa,c) and pKa,f of free protein (KPf 10pKa,f ) can be calculated
by simultaneously solving Eq. (29) for nH determined at the corresponding pH
values, even when the ligand affinity is too tight to be measured (40). In practice, nH
is determined in a series of buffers of different ionization enthalpies as a function of
pH. Equation (29) is used to determine the pKa of the protein in the free and
complexed state. With these values KB at the tight binding conditions can be
calculated by Eq. (25) (40,41). In general this treatment can be applied to more
complicated systems that involve two protons linked with binding (31,41). A similar
approach based on free energy of binding linked to the number of protons
transferred as a function of pH has been developed (39).
The power of the calorimetric approach in evaluating proton linkage lies in the
fact that H can be determined with high precision under conditions where KB is
not measurable, and thus the contributions of the linkage.
Thermodynamic Linkage to Temperature
The fundamental Eqs. (19) and (20) demonstrate that the equilibrium constant
for a process is related to the standard entropy and enthalpy changes, and to the
absolute temperature. The temperature dependence of the changes in free energy
(G) of the Gibbs-Helmholtz equation for a thermodynamic system is described as

G=T H
2 30
T T
where T is the absolute temperature and H is the reaction enthalpy. Substitution of
Eq. (30) with Eq. (19) yields the familiar vant Hoff equation:
lnKB H
31
1=T R
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where KB is the binding constant. The temperature dependence of KB is commonly

analyzed by means of the vant Hoff plot, whose basis is Eq. (31). Measuring KB over
a temperature range and plotting lnKB vs. 1/T yields the vant Hoff enthalpy
(HvH). It is calculated from the slope of the plot, according to Eq. (31).
When H is known, then integration of Eq. (31) gives the temperature
dependence of the equilibrium constant:

H 1 1
KB T KB T0 exp 32
R T T0
Equation (32) explicitly assumes that H is constant over the temperature range
TT0. It has been shown for many biological proteinligand interactions that this
assumption is not valid, with H often being temperature dependent.
The temperature dependence of the binding enthalpy, Cp, is described by

Eq. (23), and combination with Eq. (32) leads to the extended form of the vant Hoff
equation that accounts for the temperature dependence of H:

H T0 1 1 Cp T T0
KB T KB T0 exp ln 1 33
R T T0 R T0 T
where KB (T) is the binding constant to be calculated at temperature T, KB (T0) is
the binding constant experimentally determined at temperature T0, and H(T0) is
the experimental enthalpy change at temperature T0.
In cases where the affinity is too tight to be measured directly by ITC under
ambient conditions, the binding constant becomes accessible by changing to a
temperature whereby binding is lowered. After determining H and the temperature
dependence thereof, the binding constant can easily be calculated for the
temperature of interest (42).
Displacement Experiments
As an alternative approach for measuring tight binding constants, a displace-

ment experiment can be carried out. The protein of interest is presaturated with a
more weakly binding ligand whose binding parameter can be determined directly,
and this ligand is displaced by injecting a ligand that binds more strongly. The first
ligand will compete with the second and thus reduce the apparent binding constant
(4346).
A first experiment yields the thermodynamic parameters for the first ligand A
(H1, K1), the second titration gives apparent values for the second ligand B
(H2obs , K2obs ). The observed association constant for binding of B K2obs in the
presence of A is given by:
K2
K2obs 34
1 K1 A
The observed binding enthalpy (H2obs ) is given by:
K1 A
H2obs H2 H1 35
1 K1 A
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19
Thus, from knowledge of H2obs and K2obs the affinity K2 and binding heat H2
for the tight binding ligand B can be deduced using Eqs. (34) and (35). Both
equations assume that the concentration of B does not change during the
measurment. This can be achieved when B is also present in the syringe solution.
However, it is also possible to include dilution effects into the fitting procedure. An
exact mathematical treatment for the analysis of competition ligand binding using
displacement ITC has recently been published (47).
A restriction to the displacement method occurs when both ligands bind with
exothermic H, as the tight binder will be reduced by the endothermic contribution
of the dissociation of the first ligand. Therefore, it is necessary that the interaction
enthalpies for both ligands differ significantly.
INFORMATIONAL CONTENT OF ITC DATA
The gain of knowledge through thermodynamic data of binding reactions is

large and can have a dramatic impact on characterizing the molecular mechanism of
binding. The thermodynamic profile of an interaction process reflects various types
of forces that drive binding, including enthalpic contributions of bond formation,
entropic effects such as restrictions of degrees of freedom, the release and uptake of
water and ion molecules, the burial of water-accessible surface area and changes in
vibrational content.
As outlined before, calorimetry measures a global property of a system, and thus
reflects the sum of all concomitant phenomena which must be carefully analyzed and
quantified in order to yield parameters conforming to the binding event properly.
Moreover, the quality of the deconvoluted parameters depends on the appropriate
model used. Nevertheless, once reliable data are available, the informational content
of thermodynamic data can have an intriguing impact on the characterization of
molecular mechanisms of binding.
Binding Free Energy
The Gibbs free energy of binding is the most important thermodynamic

description of binding, since it determines the stability of any given biological
complex, and it has been (and still is) a useful analytical tool for the phenom-
enological characterization of structurefunction relationships. The typical analysis
of calorimetric data involves fitting an appropriate model to the data, i.e., simple
single-site or two-site binding model, which yields the binding constant. But often,
the system under investigation exhibits a more complex behavior and more
sophisticated models must be applied (multiple interacting-site models). The
macromolecule may undergo ligand-induced changes, be they conformational
adaptations in the binding site or self-association of the receptor, which will
contribute to the total free energy of binding. These effects should be evaluated
independently with companion methods, i.e., analytical ultracentrifugation,
analytical gel filtration chromatography, and spectroscopic methods.
ORDER REPRINTS
In several recent publications it has been proposed to dissect binding free energy
into several contributing terms. The total binding free energy contains a contribution
typically associated with the formation of secondary and tertiary structure (van der
Waals interactions, hydrogen bonding, hydration, conformational entropy),
electrostatic and ionization effects, contributions due to conformational transitions,
loss of translational and rotational degrees of freedom, and others that must
be accounted for on an individual basis (4855). For example, an observed G can
be the same for, an interaction with positive S and H (binding dominated by
hydrophobic effect) and an interaction with negative S and H (when specific
interactions dominate). Moreover, interacting systems tend to compensate enthalpic
and entropic contributions to G, making binding free energy relative insensitive to
changes in the molecular details of the interactions process (5557). Thus,

consideration of H and S are crucial for a detailed understanding of the free
energy of binding.
Binding Enthalpy
The observed heat effect of a binding reaction is a global property of the whole
system under investigation, reflecting the total heat change in the calorimetric cell
upon addition of ligand. On the one hand, the measured heat contains contribution
from unspecific heat effects, on the other hand, there might be protonation effects
coupled to binding. Therefore it is of outmost importance to determine possible
contributions to the intrinsic binding enthalpy and to correct for them. But even
corrected heat changes are composed of different contributions, which is the reason
why the enthalpy is an apparent or observed (Hobs) quantity (29).
At first appearance, the physical meaning of H seems to be simple: it
represents the changes in noncovalent bond energy occurring during the interaction.
This interpretation is too simple to describe observed H values. The measured
enthalpy must be the result of the formation and breaking of many individual bonds,
since it is barely conceivable to form bonds without breaking any others, especially
in aqueous medium. The enthalpy change of binding reflects the loss of protein
solvent hydrogen bonds and van der Waals interactions, formation of proteinligand
bonds, salt bridges and van der Waals contacts, and solvent reorganization near
protein surfaces. These individual components may produce either favorable or
unfavorable contributions, and the resultant is likely to be smaller than the specific
interactions (30).
From calorimetric studies carried out in water and D2O it was concluded that a
large part of the observed enthalpy change is due to bulk hydration effect (58,59).
Often, water molecules are placed in the complex interface, improving the
complementarity of the complex surfaces and extending H-bond networks. This
can make enthalpies more favorable, but is often counterbalanced by an entropic
penalty (6062). The role of interfacial water was directly examined by lowering
water activity by means of glycerol or other osmolytes. Complexes with a low degree
of surface complementarity and no change in hydration are tolerant to osmotic
pressure (25,6366).
ORDER REPRINTS
21
Besides the unspecific hydration effects, all direct noncovalent bonds at the
binding interface contribute to H, actually reflecting the binding enthalpy in a
strict sense. The dissection of each noncovalent interaction is very difficult since the
net heat effect of a particular bond is the balance between the reaction enthalpy of
the ligand to the macromolecule and to the solvent. Moreover, structural alterations
at the binding site due to the binding event may contribute to the binding enthalpy.
Several mutational approaches have been applied to investigate the energetics of
individual bonds: alanine scanning mutagenesis (67), removal of particular H-bonds
at the active site (68), construction of double mutant cycles (69). However, all these
approaches suffer from the problem that a direct relation between the change in H
and the removal of the corresponding specific contact in the active site cannot be
made a priori. On a theoretical basis it has been argued that decomposition of H is

not possible (70), but others favor a dissection into specific contributions (71,72).
Binding Entropy
The entropy of binding is directly calculated from G and H according to

Eq. (20). In general, it represents all other positive and negative driving forces that
contribute to the free energy. Recently, it has been proposed that the total entropy
change associated with binding can be expressed as the sum of several contributing
effects. The main factor contributing to S of complex formation is due to
hydration effects. Since the entropy of hydration of polar and apolar groups is large,
the burial of water-accessible surface area on binding results in solvent release which
contributes often large and positive to the total entropy of interaction. Another
important, though unfavorable contribution reflects the reduction of rotational
degrees of freedom around torsion angles of protein and ligand side-chains. An
additional entropy term accounts for the reduction of the number of particles in
solution and their degrees of freedom (7375).
A negative entropy change can entail different contributions, and it does not
necessarily indicate increased or unchanged hydration interfaces, but a positive
entropy change is a strong indication that water molecules have been released from
the complex surface (76).
Heat Capacity Changes
If H is determined at a range of temperatures (modern ITC instruments allow

measurements between 2 and 80 C), the change in the constant pressure heat
capacity (Cp) for an interaction is given by the slope of the linear regression
analysis of Hobs plotted vs. temperature. Often Cp does not depend on
temperature within the small physiological temperature range, although several
publications reported weak or strong dependencies (37,38).
For the binding reaction, Cp is almost always negative when the complexed
state of the macromolecule is taken as the reference state. Its origin lies in the fact
that there is strong correlation between Cp and the surface area buried on forming
a complex (7781). It has been shown that the removal of protein surface area from
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the contact with solvent results in a large negative Cp. The basis of this observation
is the different behavior of solvent on the surface of a macromolecule and to that in
the bulk, particularly with respect to water molecules interacting with hydrophobic
surfaces. This means that for any process, in which water is released from the
surface, Cp will be substantial and would be proportional to the amount of surface
involved. Through this correlation of Cp and burial of surface area, the heat
capacity provides a link between thermodynamic data and structural information of
macromolecules. Therefore in the last years, there has been considerable progress
in the parameterization of all thermodynamic parameters and the predictions
thereof (82,83).
EnthalpyEntropy Compensation
In most thermodynamic binding studies of biological systems, the phenomenon

of enthalpyentropy compensation has been described (56,57). It is characterized by
the linear relationship between the change in enthalpy and the change in entropy,
i.e., favorable changes in binding enthalpy are compensated by opposite changes in
entropy and vice versa, resulting in small changes in binding affinity over a range of
temperature. It is assumed that enthalpyentropy compensation is connected to the
properties of the solvent (water), and it appears also to be a general consequence of
perturbing weak intermolecular interactions (84,85). This is in agreement with the
fact that increased bonding in a binding process, resulting in more negative H, will
be at the expense of increased order, leading to more negative S. As both H and
S are connected to Cp, it is not surprising that both parameters are correlated.
In terms of medicinal chemistry and rational drug design, this phenomenon is a
difficult problem to overcome in order to increase binding affinity of a compound to
the protein of interest. The ideal optimization strategy requires the implementation
of enthalpic or entropic contributions that result in a minimal entropic or enthalpic
penalty. This will induce the largest change in G, thereby defeating the deleterious
effects of enthalpyentropy compensation at the thermodynamic level.
Binding Stoichiometry
The determination of binding stoichiometries (n) is of central importance for the

characterization of binding mechanisms of biological macromolecules. ITC has
emerged as an important tool, since it enables high-precision analysis with high
reproducibility because of the computer-controlled injection of definite volumes.
Assuming that the concentrations of both interacting species are known, the binding
stoichiometry can be determined from the molar ratio of the interacting species at
the equivalence point. During fitting procedures the parameter n can either be fixed
as equal to the number binding sites per macromolecule, or it can be treated as an
additional floating parameter that is determined by iterative fitting. There are a
number of possible sources which lead to deviation from the expected values of n,
i.e., high experimental uncertainty of the data set, error in concentration of either the
ligand or the macromolecule, unspecific binding, degradation of ligand, and low
ORDER REPRINTS
23
protein quality (unfolded, missfolded). If these systematic errors can be ruled out
and the fitted values for n still deviate significantly from expected values known from
additional independent information, they should be reexamined. It has been
proposed to fit the data to the hyperbolic equation and then to reanalyze them by
means of a double reciprocal plot (30). However, it is recommended to corroborate
stoichiometric data obtained by microcalorimetry with additional independent
information.
Once the model is verified, ITC can be used as an excellent quality control tool
for the analysis of the fractional binding activity of different lots of protein (e.g.,
antibodies), for stability testing (freeze-thaw) and many more (86).
PREDICTION OF BINDING ENERGETICS
A long-standing goal of biophysical chemistry is the prediction of binding

energetics from the 3D-structure of proteinligand complexes, and it is a key element
in the field of structure based drug design. The rapidly increasing availability of high-
resolution protein structures from X-ray crystallography and nuclear magnetic
resonance (NMR) opened the field to combine structural information with
thermodynamic data of the binding process.
In the past few years, considerable progress has been made in characterizing
molecular aspects of proteinligand interactions by means of thermodynamic data.
It has been shown that the major polar and apolar contributions to the enthalpy,
entropy and heat capacity changes for protein folding and unfolding can be
described in terms of changes in solvent-accessible polar and apolar surface area
(77,80,81,8791).
The empirical parameterization based on calculation of changes in solvent
accessible surface areas was first applied to the prediction of protein folding
energetics (77,80,81,87,89,91). Since the atomic interactions involved in associations
reactions are similar (92), it was suggested to apply this approach to proteinprotein
interactions, peptide binding to proteins, and to small ligand binding. The
parameterization has now reached the state in which accurate prediction of protein
folding energetics and binding energetics is possible (73,79,93,94).
Solvent-Accessible Surface Areas
According to Lee and Richards (95), the solvent-accessible surface area (ASA) is
defined as the surface traced out by the center of a solvent probe (frequently taken as
a sphere with a radius of 1.4 A) as it moves over the surface of the protein. There are
a number of implementations described and used to determine ASA (95,96), and it is
very important to recognize that each implementation yields slightly different results.
The original description of the predicting parameters is based on the Lee and
Richards algorithm as implemented in the program ACCESS, using a probe radius
of 1.4 A and a slice width of 0.25 A, but there exist reparameterized values for other
implementations (82). When performing calculations, it must be assured that the
appropriate parameters are used, because they are dependent on the algorithm used.
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Calculation of Thermodynamic Parameters
In general, changes in solvent-accessible surface area (ASA) are determined as

the difference of ASA of the final state and of the initial state. For a molecular
interaction process, this is the difference between the ASA of the complex and the
sum of the ASA of the macromolecule and the ligand, resulting in negative values of
ASA. ASA is further subdivided into nonpolar and polar contributions by
simply defining which atoms take part in the surface. Oxygen, nitrogen and sulfur
are treated as polar and all carbon atoms as apolar. If structured water molecules
take part in the binding interface, they must be accounted for, since their presence
will contribute to the amount and type of surface area buried (82,97).
In the ideal case, structural information will be available for the complex and for
both interacting species, i.e., the free macromolecule and the ligand. This will
account for any structurally defined conformational differences that occur on
binding. If there are no conformational changes linked with the association (rigid
body binding), it is good practice to extract the free state of the protein by removing
the coordinates of the ligand from the complex. However, the assumption of rigid
body binding must be verified by other independent methods because any structural
rearrangement will contribute to the energetics. For the case where binding is linked
to order/disorder of particular regions which are not defined in the coordinate file of
the macromolecule, it may be necessary to add a model of the region. This is
especially found in protein peptide interactions, for which it is necessary to define
a solution structure of the free peptide (73,79).
Calculation of DCp
The largest contribution to the Cp for a binding process arises from
dehydration of protein and ligand surface with negative contributions due to
burial of apolar surfaces (ASAnp) and positive contributions due to burial of polar
surfaces (ASAp). From studies of the dissolution of solid model compounds,
the following relationship has been proposed (77,80):
Cp cnp ASAnp cp ASAp 36
The parameters cnp and cp that are suitable for calculations based on ASAs
determined by different programs are given in Table 1. Equation (36) is used to
predict the Cp for ligand binding.
Calculation of DH
The change in enthalpy of binding reflects the loss of proteinsolvent hydrogen

bonds, van der Waals interactions, and formation of proteinligand bonds, salt
bridges and van der Waals contacts, and solvent reorganization near protein
surfaces. H is calculated with reference to the temperature at which the apolar
contribution is assumed to be zero TH . The value of TH is the enthalpy convergence
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25
Table 1. Empirical parameters for calculation of Cp and H for three different
implementations (units for c in cal K1 (molA2)1; for h in cal (molA2)1; probe radius
1.4 A; slice width 0.25 A).a
ACCESS ACCESS NACCESS

Parameter (Presnell)b (Richards)c (Hubbard)d
cap 0.45 0.36 0.43

cp 0.26 0.25 0.26
hap 8.43 3.63 7.26
hp 31.29 23.75 29.14
hap(25) 7.35e
hp(25) 31.06e
a
Values from Ref. (82)
b
Ref. (141)
c
Ref. (95)
d
Ref. (96)
e
Ref. (97).
temperature, which is obtained from protein unfolding studies and is determined to

be 100 C (373 K) (98). Thus the enthalpy change is calculated from:

H H Cp T TH 37

where H is the polar contribution to H at TH The value for H is directly
proportional to the burial of polar surface area (77,80) and is described by
H 356 ASAp 38
where ASAp is the change in polar accessible surface area and is negative for a
binding reaction.
Above dissection contains a linear extrapolation of protein unfolding enthalpies
as a function of polar and apolar surface area (77,80,91). A regression analysis at the
medium unfolding temperature of proteins (60 C) minimizes the extrapolation
error and yields the elementary contributions per A2 of apolar (hap) and polar
(hp) surface to the enthalpy function at the reference temperature of 60 C
(Hbind(60 C)):
Hbind 60 C hap ASA hp ASAp 39
Values for hap and hp are given in Table 1. At any other temperature T,
H(T) is given by the standard equation
Hbind T Hbind 60 C CpT 333:15 40
Above calculations were shown to hold reasonably well at predicting the binding
enthalpy for proteinprotein and proteinpeptide interactions, but correlation of
structure and enthalpy has yielded inconsistent results for interactions of small
molecules (MW < 800) with biological macromolecules. A first attempt for empirical
parameterization of H for small ligands has been published recently (97). As a first
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approximation the experimental binding heat (Hexp) can be considered as the

combination of atleast three terms:
Hexp Hint Hconf Hprot 41
The intrinsic enthalpy (Hint) reflects the interaction of the ligand with the
protein and corresponds to the situation when ligand and protein adopt the same
conformation in the free and the bound state. These contributions to the enthalpy
are assumed to scale with changes in accessible surface area that can be param-
eterized. Contributions from conformational changes (Hconf) cannot be easily
described in terms of ASA. Hprot contains binding enthalpy that is due to
protonation effects that can and need to be dissected experimentally by performing
experiments in buffers of different ionization enthalpies. Once corrected for Hprot,

the calculated Hbind at 25 C, the temperature at which most binding studies
are performed, will be the sum of the constant term Hint and a term that is specific
for each ligand:
Hbind 25 Hconf 25 hap25 ASA hp25 ASAp 42
The intrinsic enthalpy is now described in terms of ASA with hap(25) and
hp(25) as the scaling coefficients that yield the elementary contributions per A2
of apolar and polar surface, respectively. The values for hap(25) and hp(25) are
given in Table 1.
In practice, a system under investigation will be analyzed using the scaling
parameters, ASA and leaving Hconf as the only adjustable parameter that needs
to be determined for each protein separately from a training set of known protein
ligand structures. This new approach is based on a HIV-1 protease database and
has been validated by six different datasets for which structural data of complexed
and free protein was available (97).
Calculation of DS
The total entropic contributions associated with binding reactions can be

expressed as the sum of three terms (75):
Stot Ssolv Sconf Sr=t 43
where Ssolv describes the change in entropy resulting from solvent release upon
binding, Sconf is a configurational term reflecting the reduction of rotational
degrees of freedom around torsion angles of protein and ligand. Sr/t entails the loss
of translational and rotational degrees of freedom when a complex is formed from
two molecules free in solution.
The most important contribution to the entropy change arises from the
solvation term (Ssolv), primarily due to burial of apolar surface area and is
approximated for any temperature (T ) by following equation:

Ssolv Cp ln T=TS 44
ORDER REPRINTS
27
where TS is the temperature at which there is no solvent contribution to the

hydrophobic entropy change and is equal to 112 C (385 K) (74,99,100).
The translational/rotational entropy term (Sr/t) accounts for the reduction of
the number of particles in solution and their degrees of freedom. Very different
estimates of the magnitude of Sr/t have been discussed (73,101). Empirical and
theoretical considerations have suggested that this term contributes 4 to 10
cal K1 mol1 to the overall entropy of a bimolecular binding event (74,102,103).
This value is numerically close to the cratic entropy of 8 cal K1 mol1(104) even if
there is no sound physical reason to equate translational/rotational entropy to the
statistical part of the mixing entropy (105).
Finally, the configurational entropy Sconf reflects contributions from changes
in side-chain conformational entropy as well as all other structural rearrangement of

protein and ligand induced by complex formation. Since Stot is experimentally
accessible, and Ssolv and Sr/t can be estimated, Sconf is calculated from:
Sconf Stot Ssolv Sr=t 45
The contribution to S from side chains involved in binding can be estimated by

considering an average contribution of 4.3 cal K1 mol1 per residue to Sconf
(73,100,107). On the assumption that only minor configurational entropic
contributions of ligand occur, a rough estimate of the number of amino acids
(Xres) participating in the interaction is available by
Sconf
Xres 46
4:3 cal K1 mol1
If the amino acid side chains directly participating in the interaction process
are known from theoretical or experimental studies, it is possible to calculate
each contribution to Sconf as the sum over the amino acids involved in binding
(108111). A binding process involves two contributions to Sconf : (i) restrictions
around side-chain torsion angles and (ii) immobilization of the peptide backbone. If
binding is not involved in order/disorder transitions, only the side-chain component
applies. The side-chain contribution is then calculated by assuming that the entropy
is zero when fully buried and scales linearly with ASA, resulting in a maximum value
when fully exposed. By applying this model, a term Sbu!ex, i must be introduced to
account for the buried-to-exposed entropy gain which differs for each side chain.
The contribution is then calculated as
X ASAsc;i
Sconf Sbu!ex;i 47
i
ASAAXA;i
where ASAsc,i is the change in ASA of side chain i on binding, and ASAAXA,i is
the ASA of the side chain in an extended Ala-X-Ala tripeptide (82). The summation
is carried out over all side chains participating in the interface. Values for ASAsc,i
are determined from structural data, estimates for ASAAXA,i and Sbu!ex,i (111)
are available, and they have been adapted for different implementations (82).
These data are presented in Table 2.
For interaction processes involving transitions, two additional terms associated
with the backbone entropy (Sbb) and with the entropy of the transition from
ORDER REPRINTS
Table 2. Total side-chain ASA (ASAsc) of Ala-X-Ala tripeptides used in parameterization

for three ASA implementations (units in A2), and side-chain and backbone conformational
entropy values (Sbu!ex, Sbb, Sex!u; units in cal K1 mol1).a
ACCESS ACCESS NACCESS

(Presnell)b (Richards)c (Hubbard)d
ASAsc ASAsc ASAsc Sbu!ex Sbb Sex!u
Ala 52.1 60.4 54.6 0.00 4.11 0.00

Arg 187.9 210.2 199.6 7.10 3.39 0.84
Asn 113.8 123.0 112.9 3.30 3.39 2.25
Asp 102.4 106.5 99.4 2.01 3.39 2.15
Cyse 70.8 69.1 70.3 3.56 3.39 0.62
Cysf 91.9 94.5 92.0 3.56 3.39 0.62

Gln 128.7 142.8 122.3 5.02 3.39 2.13
Glu 117.5 135.8 124.5 3.54 3.39 2.27
Gly 0.0 0.0 0.0 0.00 6.50 0.00
His 144.3 152.2 144.9 3.44 3.39 0.79
Ile 123.8 138.5 135.9 1.74 2.17 0.67
Leu 134.5 150.6 143.8 1.62 3.39 0.24
Lys 156.9 177.7 155.6 5.85 3.39 1.03
Met 158.5 160.8 158.0 4.54 3.39 0.57
Phe 176.7 179.4 172.0 1.41 3.39 2.89
Pro 90.7 104.8 90.7
Ser 68.6 76.6 71.7 3.68 3.39 0.55
Thr 105.3 112.1 105.4 3.30 3.39 0.48
Trp 222.7 218.7 222.4 2.75 3.39 1.15
Tyr 188.5 196.4 190.2 2.77 3.39 3.13
Val 105.6 118.0 105.6 0.12 2.17 1.29
a
Values from Ref. (82)
b
Ref. (141)
c
Ref. (95)
d
Ref. (96)
e
Values for disulfide-bonded cystine;
f
Values for free cysteine.
the exposed/folded state to the exposed/unfolded state (Sex!u) must be included

(82). Estimates of these contributions are also available (110), and are summarized
in Table 2.
For nonpeptide ligands a special empirical parameterization has been proposed
to account for changes in conformational degrees of freedom between free and
complexed forms of ligand (112). As a first approximation, it is assumed that the
conformational entropy will be proportional to the number of rotatable bonds. Since
effects from excluded volume increase with the number of atoms for a given number
of rotatable bonds, the conformational entropy change for a nonpeptide ligand
(Sconf, np) is considered to be a linear function of the number of rotatable bonds
(Nrb) and the total number of atoms (Nat):
Sconf;np k1 Nrb k2 Nat 48
ORDER REPRINTS
29
To date, Eq. (48) has not yet been widely applied, and in fact it has only been
tested for the analysis of HIV-1 protease inhibitors. However, for these interactions
k1 was found to be 1.76 cal K1 mol1, whereas k2 equals 0.414 cal K1 mol1
(83,112).
Linkage Effects
Above calculations apply for systems that do not involve linked equilibria. If
binding of a second ligand is coupled to binding of a first one, the contributions
of the second equilibrium must be considered. Protonation linkage is a common
phenomenon in interaction processes. Its contributions to the binding energetics can

be determined experimentally, using a global analysis of experimental data as a
function of pH, temperature, and buffer ionization enthalpy (40,94).
THERMODYNAMICS AND RATIONAL DRUG DESIGN
The rapidly increasing availability of high-resolution protein structures has

opened the possibility to use structural information in the design of new drugs. The
central problem of structure-based design studies is the understanding of the features
dictating the energetics of the interaction of a ligand with a macromolecule, i.e., the
accurate prediction of the Gibbs free energy that determines the binding affinity.
The Thermodynamic Approach
The prediction of binding energetics is greatly complicated by the effects of

enthalpyentropy compensation (84,85) which means that an increase in H does
not contribute to the binding affinity, as the improvement is only achieved by a
compensation cost in the TS term. The sheer number of entropic and enthalpic
effects contributing to the observed G makes it difficult to rationalize and predict
binding affinities. Additional energetic effects will arise from any differences in
ligand conformations in the free state in solution and bound to the macromolecule.
Moreover, water molecules play an important role in adapting the binding pocket
to different ligands.
The strength of the thermodynamic approach is that it has become possible to
dissect the contributing forces (H, S, Cp) which make up the free binding
energy of the interaction by direct calorimetric measurements, yielding effective
energetic contributions, including all interactions that are found in the system
(protein, ligand, and solvent interactions).
Current Status of the Thermodynamic Approach
Until recently, thermodynamic data have not played an important role

in molecular design, since no theoretical framework relating structural and
ORDER REPRINTS
thermodynamic data has been established. The situation has changed with the
realization that changes in solvent-accessible surface area (ASA) are related to the
thermodynamic parameters. The semi-empirically derived set of structural
energetic parameters (summarized in Table 1), based on the parameterization of
G, H, S, and Cp in terms of ASA, is a promising thermodynamic approach
to drug design.
It is possible now to predict the energetics of folding of a globular protein
(77,80,81,87,89,91) with an accuracy within 912% (82). The parameterization has
reached the state in which accurate predictions of binding energetics is possible as
well, as shown for peptideprotein and proteinprotein association (73,79,93,94),
and also for nonpeptide ligandprotein interactions (112). However, the thermo-
dynamic approach is still in its infancy, as there are no comprehensive reports

documenting the design of a high-affinity drug using a thermodynamic-directed
rational drug-design approach.
Furthermore, current parameterization is still based on protein unfolding data,
and there is need for more thermodynamic and structural characterization of
proteinligand interactions, including not only H, S, and Cp, but also
thorough investigation of phenomena linked to binding, i.e., protonation, ion
binding, and conformational changes. As the research in this field makes progress,
the structural thermodynamic database is expanded and the set of energetic
parameters can be refined to the point that will allow accurate prediction of effects of
small molecular changes. Thus, this will allow a much easier assessment of better
binding drugs.
CASE STUDIES FOR PROTEINLIGAND

INTERACTIONS STUDIED BY ITC
The term proteinligand interactions stands for the association of a

biological macromolecule (a protein) with any molecule (the ligand). This is an
arbitrary restriction to biological system and does not mean that other binding
partners do not exist or cannot be measured. As mentioned before, any binding
event that will produce sufficient binding heat will be amenable to the calorimetric
approach. This has been shown for a variety of interacting systems (for a list see
http://www.calorimetry.com) that are not subject of the present discussion.
Whereas the biological systems under investigation deal with proteins as one
binding partner, e.g., antibodies, enzymes, or receptors (soluble and membrane
bound), the ligand will include all possible interaction partners for the protein.
These can be proteins, peptides, DNA, carbohydrates, lipids, metal ions, inhibitors,
substrates, or cofactors. At this point we describe three representative examples for
biological systems, i.e., enzymes, soluble receptor moieties, and membrane-bound
receptors that have been investigated using the direct thermodynamic approach.
This will give a deeper understanding to the reader with respect to the
informational content, possibilities, and potential drawbacks for the calorimetric
experiments.
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31
Substrate Binding to HSV1 Thymidine Kinase
Introduction
An example of high medicinal interest where thermodynamic information is

essential is thymidine kinase from Herpes simplex virus type-1 (HSV1 TK). The
structure of this enzyme is known at high resolution in complex with a series of
ligands, including various substrates (natural and non-natural) and inhibitors
(113117).
Thymidine kinases (EC 2.7.1.21) catalyze the phosphorylation of dT to
thymidine monophosphate (dTMP) in presence of magnesium ions by transferring
the -phosphate group of adenosine triphosphate (ATP) to the 50 -OH group of dT.
Herpes viruses encode their own thymidine kinases, which differ considerably from
the enzyme of the human cellular host (hTK1). While the human enzyme is highly
specific, HSV1 TK is a multifunctional enzyme of broad substrate specificity and
requires low stereo-chemical specificity. Therapeutic applications involving
HSV1 TK make use of the broad substrate diversity of the viral enzyme in the
background of strict substrate selectivity of the host cell enzyme. Therefore, a
detailed thermodynamic analysis of substrate binding to the viral kinase is a
prerequisite for the successful design of new therapeutically useful compounds. ITC
has been used to investigate the thermodynamic parameters of the binding
of thymidine (dT) and adenosine triphosphate (ATP) to wild type and mutated
HSV1 TK (118,119).
Thermodynamic Parameters for dT and ATP Binding to HSV1 TK
In the ternary complex TK:dT:ATP, the substrate dT and the cofactor ATP are
located in separate and well defined binding pockets of the enzyme. Formation of the
ternary enzymesubstrate complex may either proceed through an obligatory
sequential pathway or by a random mechanism. Two sequential pathways are
possible: TK!TK:dT!TK:dT:ATP (reactions (i) and (ii) of Fig. 5), or TK! TK:
ATP!TK:dT:ATP (reactions (iii) and (iv) of Fig. 5). In a random mechanism,
TK TK:dT
(i)
(iii) (ii)
(iv)
TK:ATP TK:dT:ATP
Figure 5. Formation of the ternary enzymesubstrate complex TK:dT:ATP. The two ordered
sequential pathways are (i), plus (ii) and (iii), plus (iv), respectively. In a random binding
mechanism, all four reactions will take place.
ORDER REPRINTS
binding of one substrate is not a prerequisite for binding of the other, and all four
reactions of Fig. 5 can take place. To distinguish between ordered and random
binding, HSV1 TK was titrated with dT and with ATP, respectively. The titration
with dT was characterized by a significant exothermic heat effect. The nonlinear least
squares fit for a single-site binding model gave a KB of 1.9 105 M1 and Hbind
of 19.1kcal mol1 (Table 3). Under identical conditions ATP did not bind to empty
HSV1 TK. An ordered binding mechanism was confirmed by titrating the preformed
TK:dT binary complex with ATP (reaction ii of Fig. 5). The reaction was again
exothermic and yielded a KB of 3.9 106 M1 and Hbind 13.8 kcal mol1.
Moreover, titration of TK with a 1:1 mixture of dT and ATP yielded within error a
heat change corresponding to the sum of the heat changes for reactions (i) and (ii) of
Fig. 5. The ITC experiment provides the binding constant KB for a single-site
reaction, and G of reactions (i) and (ii) were calculated from Eq. (19). S was
obtained from Eq. (20). Reactions (i) and (ii) were driven by favorable negative
changes in binding enthalpy and strongly opposed by unfavorable entropic
contributions. Although the reaction with the 1:1 mixture of dT and ATP was
more complex, it could still be treated as a single-site reaction if one considered
dT ATP as one ligand. ITC measurements performed in the temperature range of
1025 C showed strong dependence of H and TS on temperature while G was
almost insensitive due to enthalpyentropy compensation. Values of Cp were
calculated from the slopes of the regression lines of Hbind vs. temperature. Binding
of dT to the free enzyme was characterized by Cp of 360 cal K1 mol1. For ATP
binding to the TK:dT complex, Cp of 140 cal K1 mol1 was determined,
and for the titration of the enzyme with a 1:1 mixture of dT and ATP, a Cp
Table 3. Thermodynamic parameters for the binding of thymidine and ATP to HSV1 TK
at pH 7.5 and 25 C.ab Values of H are corrected for protonation effects by Eq. (6).
TK dT (reaction (i)) TK:dT ATP (reaction (ii)) TK dT/ATPc

Temp.
( C) H G TS H G TS H G d TS
10 13.6 7.1 6.5 11.7 8.9 2.8 25.4 18.9 6.5

15 15.8 7.0 8.8 12.1 8.9 3.2 27.9 18.9 9.0
20 17.4 7.1 10.2 13.0 9.1 3.9 30.5 17.2 13.3
25 19.1 7.2 11.9 13.8 9.0 4.8 33.1 16.8 16.3
Cp 0.36 0.14 0.51
a
Values of G, H, and TS in kcal mol1, Cp in kcal K1 mol1.
b
Values are the mean of triplicates. G was calculated from G RT lnKB, where KB is the
binding constant determined by ITC. Uncertainty of G is within 0.35 kcal mol1 of the
mean. Errors of H are about 5% and mainly reflect the error in ligand concentration.
Maximal possible errors of TS are 1.5 kcal mol1. Errors of Cp were estimated by
reduction of the data set by one data point at a time and were, on average, 0.02
kcal K1 mol1, i.e., within 515% of the reported mean.
c
1:1 mixture of dT and ATP.
d
Calculated from G RT ln(KB)2.
ORDER REPRINTS
33
of 510 kcal K1 mol1 was measured. The latter value was very close to the sum
of Cps of reactions (i) and (ii).
The stoichiometry of binding obtained from all experiments was in the range
0.70.8 mol ligand per mol of HSV1 TK monomer. Deviation from a value of 1 was
due to the presence of inactive protein (118).
Protonation Effects
Titration experiments were repeated in various buffers of different Hion. The

intrinsic enthalpy of binding, Hbind, was obtained from the intercept (Hion 0) of
a plot according to Eq. (6). Protonation/deprotonation was negligible in the case of

dT binding to the free enzyme (Hobs Hbind). An uptake of 0.31 protons was
observed with ATP binding to the TK:dT complex. Titration with the 1:1 mixture of
dT and ATP lead to the uptake of 0.35 protons. It follows that proton uptake
occurred with ATP binding but not with dT binding. Heat changes from ITC
experiments were corrected accordingly (118).
Decomposition of Entropy Changes
The results for the decomposition of Stot for substrate binding to HSV1 TK are
summarized in Table 4. It is evident that the favorable solvent contribution Ssolv is
overcompensated by the large unfavorable conformational entropy change Sconf.
The contribution by Sr/t is unfavorable but small. In accordance with a
thermodynamic cycle, the decomposed entropy changes of reactions (i) and (ii)
add up to the changes calculated for the titration of the enzyme with the 1:1 mixture
of dT and ATP.
Solvent reorganization provided a substantial gain in binding entropy due to
water release, about 90 cal K1mol1 for dT binding and about 35 cal K1mol1
for ATP binding. This considerable Ssolv is in agreement with fact that
phosphorylation of an acceptor OH-group must occur in the absence of
competing water molecules. After subtracting from Stot the favorable Ssolv
Table 4. Decomposition of entropy changes for substrate binding to HSV1 TK at 25 C.a
Reaction Stotb Ssolvc Sconfd Sr/te
TK dT, reaction (i) 39.9 92 124 8

TK:dT ATP, reaction (ii) 16.1 36 44 8
TK dT/ATP 54.7 133 172 16
a
In cal K1mol1.
b
Calculated from values of TS in Table 3.
c
Calculated from Eq. (44) using Cp from Table 3.
d
Calculated from Eq. (43).
e
Value for bimolecular and trimolecular reaction, respectively (104).
ORDER REPRINTS
term and the small unfavorable cratic Sr/t, a large unfavorable entropic
contribution Sconf remained. Sconf may have two main origins, the first being
freezing of bond rotations, i.e., amino acid side chains which are in direct
contact with the bound substrate or involved in building a closed or more
compact conformation, become less mobile. The other contribution to Sconf is
partial folding or tightening of domains, particularly in the area of substrate
binding. Interdomain movements within the enzyme dimer also can contribute to
Sconf if the HSV1 TK dimer becomes more closed in the substrate-bound form.
An estimation of these contributions according to Eq. (47) showed that
approximately 95% of Sconf may be due to conformational domain movements
and partial folding, or refolding, of domains, in line with the general concept that
nucleosidenucleotide kinases undergo conformational changes during their

catalytic cycle (120,121).
Correlation Between Cp and Surface Area Buried on Substrate Binding
The experimentally determined parameters Cp and Hbind (Table 3) were

used to simultaneously solve Eqs. (36) and (39), yielding the total area
apparently buried (ASAtot) during the binding reactions (Table 5). ASAtot
for the reaction with a 1:1 mixture of dT and ATP was 5100 A2. This change
in area corresponded very well to the sum of the surface changes for the
individual reactions (i) and (ii) (Fig. 5). These figures seem to be very large for
the binding of the two small substrate molecules and are comparable to the
surface buried on folding of a small globular protein of 5060 residues. As no
structural information is available for HSV1 TK in the ligand-free state, ASAap
Table 5. Changes of solvent accessible surface area (ASA) caused by substrate binding to
HSV1 TK.a
Calculation Reaction ASAap ASAp ASAtot Cpdcalc Cpeexp
Rigid bodyb TK dT 400 200 600 130 360

TD parametersc Reaction (i) 1750 1500 3250
Rigid bodyb TK:dT ATP 300 450 750 20 140
TD parametersc Reaction (ii) 850 850 1700
Rigid bodyb TK dT/ATPf 700 650 1350 150 510
TD parametersc 2650 2450 5100
a
ASA in A2, Cp in cal K1 mol1.
b
ASA calculated by removing dT, ATP, or both, from the crystal structure of the
TK:dT:ATP complex (113).
c
ASA calculated by simultaneously solving Eqs. (36) and (39) using experimental values of
Cp and Hbind from Table 3.
d
Calculated from Eq. (36) with ASA calculated from the crystal structure of TK:dT:ATP
(rigid body assumption).
e
From Table 3.
f
Reaction with 1:1 mixture of dT and ATP.
ORDER REPRINTS
35
and ASAp could not be tested against actual structural data. As an

approximation, ASA values were obtained for the ternary crystal complex
after removal of dT and ATP. The buried surface area calculated in this way
corresponds to a rigid body binding model in which no conformational changes
take place when dT and ATP bind. As seen in Table 5, ASAtot was very much
smaller, resulting in calculated Cp values, using Eq. (36), that were
correspondingly smaller than experimental Cp. Even so the calculated surface
area changes may be rough estimations, the significant discrepancy to a rigid
binding model is an indisputable sign for significant conformational rearrange-
ments accompanying substrate binding to HSV1 TK. Moreover it is in very good
agreement with the finding that the major unfavorable entropic contribution is
due to loss of conformational entropy.
Mutational Studies
The mechanism of the broad substrate diversity observed with HSV TK1 was
investigated by a thorough mutational study, kinetic measurements and ITC. The
residue triad H58/M128/Y172 was identified to confer distinctive binding of an
exceptionally large variety of substrates to HSV TK1 and to guide catalytic
properties. Mutations in this triad (H58L, M128F, Y172F) have been prepared
and were analyzed by ITC (Table 6).
The mutation M128F with a phenylalanine in this position resulted in a
completely inactive enzyme when examined by kinetic measurements and an HPLC
assay. It was shown that the lack of activity for the M128F mutant was not due to
hydrophobic collapse initiated by the mutation, but was the result of disturbed dT
binding (200-fold reduced affinity compared to the wild-type enzyme) that yielded an
unproductive orientation of the substrate. This view is corroborated by the strongly
reduced binding enthalpy, in combination with the more than seven-fold reduction
of unfavorable entropy. It is evident that the major structural changes needed for
catalytic competence, which are induced by dT in wild-type HSV1 TK, could not
take place.
Similar results were found with the double mutant M128F/Y172F. In this
mutant the dT binding site is formed by a double-F sandwich, a particular
Table 6. Thermodynamic data for dT binding in presence of ATP of wild type HSV1 TK and
triad H58/M128/Y172 mutants.
HSV1 TK H (kcal mol1) KB (105 M1) G (kcal mol1) TS (kcal mol1)
Wild type 26.35 0.47 229 146 9.95 0.40 16.4 0.85
M128F 8.14 0.20 1.04 0.10 6.84 0.08 1.30 0.20
M128F/Y172F 3.71 0.08 0.34 0.19 6.14 0.34 2.43 0.42
H58L/M128F/Y172F 19.82 0.13 2.61 0.06 7.38 0.01 12.44 0.14
ORDER REPRINTS
combination that also occurs in various thymidine kinases. In this case even the sign
for the entropy changed, giving evidence for a favorable preformed binding pocket
with a subsequent reduced induced fit movement. However, the hydrogen bonding
network seems to be impaired as deduced from the almost 700-fold reduced dT
affinity as compared to wild-type HSV1 TK.
Sequence alignments show that this double-F combination never occurs with a
histidine at position 58 (HSV1 TK numbering). Subsequently H58 was replaced by
leucine, giving the triple mutant H58L/M128F/Y172F. With this third mutation
enzymatic activity was regained, but the broad substrate diversity was lost since
activity and kinetic studies as well as HPLC assays did not show any activity of the
triple mutant against non-natural substrates. Although the binding affinity is almost
90-fold reduced, binding enthalpy and entropy adopt similar values as for wild-type
HSV1 TK, indicating restored flexibility of the enzyme.
Conclusion
This is the first report providing a comprehensive thermodynamic description

of substrate and cofactor binding to HSV TK1, a representative of the large family
of nucleotide and nucleoside kinases. The results obtained by titration micro-
calorimetry reveal an extreme case of positive heterotropic interaction. Formation
of a binary complex of thymidine with HSV1 TK is a stringent prerequisite for
ATP binding. Since the ATP binding site is in fact generated by thymidine binding,
one expects the enzyme to undergo considerable conformational rearrangements.
This has been supported by a semi-empirical analysis of the observed heat capacity
and entropy changes, which were large and negative and indicated burial of
molecular surface to an extent much larger than expected if the substrate binding
sites would pre-exist on the apo-enzyme and no rearrangement would occur (rigid
body binding model). The favorable gain in entropy from water release from buried
surface was overcompensated by a large decrease in conformational entropy. The
findings support the view that substrate binding to HSV1 TK leads to a
conformational closing of the substrate binding sites to bring thymidine and ATP
into an orientation appropriate for catalysis. Further mutational studies in the
residue triad H58/M128/Y172 supported this view and could shed light on possible
mechanisms important for substrate diversity. Nevertheless, the details of the
predicted rearrangements have to await a firm structural foundation. The direct
thermodynamic approach using ITC proved to be an invaluable tool for this
study. It made not only possible to characterize HSV1 TK interactions with its
substrate and cofactor thermodynamically, but it was also an invaluable tool
to investigate mutants that were not amenable through standard kinetic measure-
ments. The lack of observable activity does not necessarily mean that binding is
prevented, but the association can be altered either subtly or dramatically, thus
leading to catalytic incompetence. ITC clearly is the method of choice for studies of
that kind.
ORDER REPRINTS
37
Specificity of Citrate Binding to the Histidine Autokinase CitA Receptor
Introduction
The periplasmic receptor domain of the sensor kinase CitA of Klebsiella

pneumoniae represents another very interesting biological system that has been
investigated by the direct thermodynamic approach (122). In bacteria, regulation
of gene expression in response to changing environmental conditions is often
accomplished by two-component regulatory systems. In their simplest form they
consist of two modular proteins, the sensor kinase and the response regulator. The
sensor kinase responds to a certain stimulus by autophosphorylation of a conserved
histidine residue. Subsequently the phosphate group is transferred to a conserved

aspartate residue of the response regulator that mediates adaptations in gene
expression or cell behavior. In most cases sensor kinases are transmembrane proteins
with an extracellular N-terminal sensor domain flanked by two transmembrane
helices and a cytoplasmic C-terminal autokinase domain that is connected to the
second transmembrane helix via a linker region of variable length (123). With this
architecture bacteria are capable to sense external stimuli and transduce information
to the cytoplasm. Although many two-component systems are known, only in a few
cases have the primary stimulus of the sensor kinase been identified. In part this lack
of knowledge is due to the difficulties in isolating and purifying the membrane-
bound kinases. The strategy to overcome this problem is the expression of the
extracellular N-terminal sensor domain as a separate and soluble protein that allows
studying its binding properties.
The sensor kinase CitA of Klebsiella pneumoniae and its cognate response
regulator CitB form the paradigm of a subfamily of bacterial two-component
systems (124). It is involved in the regulation and expression of genes needed for
citrate fermentation. In a previous study it was proposed that citrate was the favorite
signal recognized by CitA (125). Attempts to demonstrate this function with an E.
coli in vivo system were not successful. Therefore, the periplasmic domain of CitA
was cloned and expressed as a separate, soluble receptor domain supplemented with
a C-terminal His6-tag (CitAPHis). Ligand specificity and thermodynamic binding
properties were investigated using ITC (122).
Citrate Binding Studies Using ITC
Isothermal titration calorimetry was applied to describe the thermodynamic

properties of citrate and citrate-analog binding to CitAPHis. Citrate binding to
CitAPHis was shown to be an exothermic process that could be fit according to a
single-site binding model, yielding a KD value (KD 1/KB) of 5.5 mM, an observed
enthalpy change (Hobs) of 18.25 kcal mol1. In contrast to citrate, neither
isocitrate nor tricarballylate exhibited demonstrable binding to CitAPHis under the
same conditions. Additional displacement experiments were necessary in order to
exclude complete entropically driven binding which would not release significant
binding heat. The results of these experiments showed that the presence of isocitrate
and tricarballylate did not alter the binding behavior of citrate towards CitAPHis.
ORDER REPRINTS
Therefore it was concluded that both isocitrate and tricarballylate do not bind to the
citrate binding site.
Investigation of the Probable Citrate Species Binding
Depending on the pH, citrate exists in four different species, i.e., H3-citrate,
H2-citrate, H-citrate2, and citrate3 with pK1 3.13, pK2 4.76, and pK3
6.40 (126). In order to determine the preferred ligand species, the pH-dependency of
the citrate binding constant to CitAPHis was determined in the range of pH 4.09.0
(Table 7). The curve obtained from a plot of log KB vs. pH showed a maximum at
about pH 5.7 and decreased above and below this value, indicating that citrate
binding is linked to ionization events (41). The Hobs and S values were negative
over the pH range studied, showing that binding of citrate to CitAPHis was driven by
the enthalpy change, whereas the entropy change was opposite.
It is very likely that this result can be explained by the assumption that
H-citrate2 is the citrate species responsible for the interaction, as the affinity of
citrate to CitAPHis at different pH values closely reflects the appearance of the
H-citrate2 form at the corresponding pH. As deduced from Table 7, the maximal
affinity was found at pH 5.7 and it decreased above and below this value, similar to
the occurrence of the divalent citrate form. An obvious explanation of this result
is that CitAPHis binds the H-citrate2 species specifically and therefore proton
release has to occur at pH values below pH 5.7 (where the H2-citrate species
becomes dominant) and proton uptake must take place at pH values above
pH 5.7 (where the citrate3 form becomes prevalent). Assuming that the H-citrate2
species is recognized by CitAPHis, four functional groups are available for binding
interactions, i.e., one uncharged and two charged carboxyl groups and the hydroxyl
group. Current crystallographic data of macromolecules involved in binding of
uncomplexed citrate clearly indicate that citrate binding is preferred in the extended
conformation with important interactions of all four functional groups (127130).
Table 7. Thermodynamic parameters of citrate binding to CitAPHis at 25 C in 50 mM

sodium phosphate buffer at different pH values as determined by ITC.a
KB KD Hobs G TS
pH n (104 M1) (mM) (kcal mol1) (kcal mol1) (kcal mol1)
4.0 0.98 0.01 6.26 1.33 16.7 3.6 24.71 0.04 6.53 0.13 18.18 0.17
5.0 0.85 0.01 38.55 1.25 2.6 0.1 23.37 0.40 7.62 0.02 15.75 0.41
6.0 0.80 0.01 51.65 2.95 1.9 0.1 20.60 0.19 7.79 0.03 12.81 0.15
7.0 0.89 0.01 18.25 0.75 5.5 0.2 18.26 0.05 7.17 0.02 11.08 0.02
8.0 0.85 0.01 9.05 0.09 11.1 0.1 17.52 0.18 6.76 0.01 10.76 0.17
9.0 0.83 0.01 6.47 0.02 15.5 0.1 17.89 0.04 6.56 0.01 11.33 0.04
a
The data represent the mean of two independent experiments. Errors are quoted as standard
deviations for the two repeats of each titration.
ORDER REPRINTS
39
Table 8. Thermodynamic parameters of citrate binding to CitAPHis at 25 C in 50 mM

sodium phosphate buffer pH 7.0 including different Mg2 concentrations as determined
by ITC.a
MgCl2 KB KD Hobs G TS

(mM) n (104 M1) (mM) (kcal mol1) (kcal mol1) (kcal mol1)
0 0.89 0.01 18.25 0.75 5.5 18.26 0.05 7.17 11.08

2 0.83 0.01 7.78 0.17 12.9 18.88 0.02 6.67 12.21
10 0.85 0.05 2.47 0.20 40.5 21.30 0.20 5.99 15.31
20 0.89 0.03 1.74 0.11 57.5 21.40 0.11 5.78 15.62
a
The data were obtained from a single experiment for each Mg2 concentration and the errors
quoted for n, KB, and H are those for the least-squares fit to the binding isotherms in the
individual titrations.
The fact that neither isocitrate nor tricarballylate did bind to CitAPHis showed the
essential role of the hydroxyl group at position C3 in the citrate molecule.
Influence of Mg2on Citrate Binding
The inhibitory influence of Mg2 ions on citrate binding observed before could
be confirmed by ITC. As shown by the titration experiments presented in Table 8,
increasing Mg2 concentrations resulted in an exponential decrease of the binding
affinity. At 20 mM Mg2, 10-fold reduction of KB was measured. In the context with
the above discussion of the four citrate species at different pH, it became clear that
the reduced binding affinity of citrate in the presence of Mg2 ions was due to Mg2
complexation by citrate3. Using a stability constant of 1585 M1(126) for the
Mgcitrate complex, the preferred Hcitrate2 species is reduced in the presence of
20 mM Mg2 at pH 7.0 to less than 0.01%, and nearly all of the citrate (96.2%) is
complexed as Mgcitrate. Crystallographic data of metal citrate complexes
generally show the hydroxyl group and either one or two of the carboxyl groups
being involved in metal binding (131). This means that the Mgcitrate complex has
to dissociate before the functional groups can interact with CitAPHis. This behavior
is reflected by the occurrence of additional binding enthalpy in the presence of Mg2.
In control experiments the additional amount of binding enthalpy (Hobs) and
entropy (S) corresponded very well to the dissociation heat and entropy of the
Mgcitrate complex. In summary, the pH and Mg2 dependency of citrate binding
to CitAPHis indicated that Hcitrate2 was the preferred binding species in a non-
restricted, extended conformation and that the inhibitory effect of Mg2 was not due
to an interaction with CitAPHis, but only due to complex formation with citrate.
Binding Stoichiometry
All data fitted excellently to a single-site binding model. Analysis of all ITC
experiments for the binding stoichiometry revealed values that varied between
ORDER REPRINTS
0.85 and 0.95 (mean value 0.87 0.06), which was in agreement with independent
binding assays using [14C]-citrate that typically gave values between 0.6 and 0.8
(122). These data clearly show that one molecule of citrate binds per CitAPHis
molecule. Thus deviations from the integer value are most likely due to inaccurate
protein determination and/or to missfolded protein.
Conclusion
In this study the direct thermodynamic approach turned out to be very powerful.
For the first time it was possible to describe the sensory characteristics of the
periplasmic domain of the sensor kinase CitA, and it could be shown that it
functions as a highly specific citrate receptor that binds citrate in a nonrestricted,
extended conformation with a 1:1 stoichiometry. Measurements at different pH
values shed light on the responsible and preferred ligand species for the receptor. The
displacement studies using the citrate analogs isocitrate and tricarballylate gave
important hints to the essential role of the hydroxyl group at C3. In addition the
inhibitory effect of Mg2 could be very well observed in the ITC experiments, and
the interpretation of the results clearly indicated that CitAPHis is not inhibited due to
a separate receptor-Mg2 interaction, but only due to complex formation with
citrate. ITC was also used to localize important amino acids involved in citrate
binding by studying the influence of point mutations in CitAPHis(132). Just recently,
the crystal structure of CitAPHis in complex with citrate has been solved (133), and
it shows that the findings learned from the thermodynamic data fit very well to
the structural data.
ProteinLigand Interactions of Membrane Bound Receptors
In this part of the review we would like to focus on biological systems that are
difficult to address: membrane bound receptors. In general, the direct thermo-
dynamic approach is also applicable to such systems as long as ligand binding is
accompanied by a significant heat change. The most severe problems to address are
the quantities of receptor that are needed, and the stability and preparation of
concentrated receptor solutions. Membrane associated proteins are intrinsically
difficult to treat and usually need special purification protocols that include
detergents, vesicles and/or liposome containing preparations. At this point it must be
emphasized that ITC instrumentation does not at all exclude the use of such
complicated or even turbid systems. As the preparation of membrane bound
receptor can be solved in a lot of cases, it remains to provide sufficient amounts of
protein. With the latest advances in recombinant DNA technology and procaryotic
as well as eucaryotic expression systems, it has become possible to prepare significant
(i.e., mg) quantities of pure receptors. This means that in the near future more
membrane receptor systems will become amenable to the direct thermodynamic
approach.
To our knowledge the first system to which ITC was applied for investigating
membrane bound proteinligand interaction, was the serine receptor of Escherichia
ORDER REPRINTS
41
coli chemotaxis (134). Two years later followed the thermodynamic work on colicin-
N binding to the trimeric membrane bound porins OmpF, OmpC, and Phoe (135).
Later in 2003 ITC was used to shed light onto human apo- and holo-transferrin
binding to the Neisseria meningitidis transferrin receptor (136). The next section is
dedicated to the most recent example published, in which the interaction
characteristics for the sensory rhodopsin II of Natronobacterium pharaonis with its
cognate transducer are examined using ITC (137). It is a particularly interesting
example for the case of lateral membrane bound receptor interaction. Some
methodological aspects will be discussed since they may serve as starting point for
the development of new projects in this field.
Interaction of Transducer Fragments with

Natronobacterium pharaonis Sensory Rhodopsin II
Introduction
Phototaxis of Natronobacterium pharaonis is mediated by the two sensory

rhodopsins SRI and SRII. They consist of seven transmembrane helices and a retinal
chromophore attached to the protein, and they share structural homologies as well
as functional properties. The first photoreceptor SRI enables the bacteria to seek
light conditions optimal for the function of the light-driven ion pumps and to avoid
ultraviolet light. The second photoreceptor SRII conveys negative phototaxis, which
might enable the bacteria to evade harmful conditions of high oxygen concentrations
in the presence of light (138). Both receptors are bound to membrane proteins
(halobacterial transducers of rhodopsins; HtrI, HtrII) that consist of a transmem-
brane two-helical domain and a coiled/coil cytoplasmic domain. SRII binds strongly
to its cognate transducer NpHtrII. Light excitation of the SRII:NpHtrII complex
leads to conformational changes in both proteins. The cytoplasmic domain of the
transducer becomes activated and in turn triggers the two-component signalling
cascade.
SRII and its cognate transducer NpHtrII form a tight 2:2 complex on
membranes, whereas in micelles it dissociates to a 1:1 homodimer (139). In order
to elucidate the dimerization and to determine the size of the receptor-binding
domain of the transducer, the dissociation constant (KD) of a series of shortened
transducers to the receptor has been analyzed by ITC (137).
Methodological Aspects
A major problem to be solved for calorimetric studies remains the availability of

sufficient quantities of the membrane bound receptor SRII. The authors of this
article used standard DNA techniques for the cloning. The gene of interest was
cloned into a pET plasmid and the receptor was expressed in BL21(DE3) as a
C-terminal His6-tagged protein. Expression took place at 37 C in 2YT medium in a
30-l fermenter. The receptor was localized in the E. coli membrane. Subsequently
crude membranes were sedimented by centrifugation and solubilized in buffer
ORDER REPRINTS
containing 1.5% n-dodecyl--d-maltopyranoside (DDM). Isolation and purification

was achieved by means of Ni-NTA agarose, but always in presence of the detergent
(0.5%). The receptor was purified with a yield of up to 1 mg/L of cell culture with a
purity of >90%. The various transducer variants are membrane-bound proteins as
well, and they were produced in a similar way affording 12 mg/L of culture.
For the ITC measurements the receptor was concentrated to 7001000 mM
(1826 mg/mL). The transducer solutions were used at 70100 mM (1.31.8 mg/mL).
Both solutions were dialyzed at 4 C overnight against degassed buffer containing
0.05% (w/v) DDM. The receptor solution (250 mL) was titrated into the solution of
the transducer placed in the cell (1.4 mL). Heat effects due to the detergent were
evaluated by control experiments using detergent-containing buffers (1% DDM
in the syringe; 0.1% DDM in the cell chamber). Titrations were carried out at
22 and 45 C in order to improve signal-to-noise ratios.
Results and Discussion
Binding affinities of the four shortened transducer fragments (NpHtrII-82,

NpHtrII-101, NpHtrII-114, NpHtrII-157) to the receptor SRII were determined
using ITC. It was necessary to measure the two longer fragments (NpHtrII-114,
NpHtrII-157) at elevated temperature (45 C) in order to detect sufficient binding
heat. Shorter fragments aggregated at higher temperature and were therefore
measured at 22 C. Both of the longer transducer fragments displayed no detectable
heat effect at lower temperature. The thermodynamic parameters of fragment
association to the receptor are shown in Table 9. The dissociation constants for
NpHtrII-157 and NpHtrII-114 were almost identical in the range of 200 nM, and the
binding heat was almost identical with 4.3 kcal mol1. However, the KD of
NpHtrII-101 was increased by almost two orders of magnitude, indicating that the
sequence between 101 and 114 is important for the receptortransducer interaction.
The shortest fragment (NpHtrII-82) was inactive in the binding assay. All three
NpHtrII fragments exhibited a stoichiometry of 1:1 within experimental error.
It must be pointed out that the data do not necessarily mirror the situation in
native membranes and the association constants and H measured might represent
minimal values. For example from the well-documented glycophorin A dimerization
Table 9. Thermodynamic parameters of the association of NpHtrII fragments to NpSRII.a
KB KD Hobs G S
NpHtrII T (K) (106 M1) (nM) (kcal mol1) (kcal mol1) (cal K1 mol1)
157 318 6.2 160 4.28 9.88 17.62

114 318 4.32 240 4.20 9.65 17.0
101 295 0.1 104 1.39 6.74 18.20
82 295 <0.01 >105
a
The measurements were performed at indicated temperatures in degassed buffer containing
150 mM NaCl, 10 mM Tris/HCl (pH 8), and 0.05% DDM.
ORDER REPRINTS
43
in membranes it could be shown that G of monomer association was influenced

by the type and concentration of the detergent used, strongly influencing the
thermodynamics of membraneprotein interactions. Despite these restrictions the
data provide important results on membraneprotein interactions and shed light on
the structure and size of the receptor-binding domain.
Conclusion
Complex formation of NpSRII and its transducer NpHtrII is a special case in

the sense that the association proceeds laterally along the membrane to form a
functional complex. Such lateral complex formation has only been elucidated in a
few examples. To date there is only rare data available that were primarily gained by
using sedimentation equilibrium and/or analytical ultracentrifugation (140). Of
general interest are those experiments that provide information about the
thermodynamics and kinetics of the transducer binding. This can be accomplished
by using a direct thermodynamic approach. As these data are difficult to determine
for membrane proteins, the present results provide a new approach to study these
interactions using a direct thermodynamic approach by means of ITC. This method
is rarely used so far for the study of membrane protein assembly due to the difficult
nature of the membrane protein family.
To our knowledge this work presents the first calorimetric approach to gain
information about the strength and energetics of lateral binding processes. This is of
general interest because it could be shown that ITC is a suitable method to study
directly membrane protein/protein interactions (137). It will contribute to our
understanding about this class of proteins, although the experiments need to be
performed in a micelle environment.
FUTURE DEVELOPMENTS
As outlined in the text and also shown with the examples, the ITC is a powerful
tool with high informational content that is applicable to various biological and non-
biological systems. A major drawback of first generation instrumentation has been
overcome by markedly increasing the sensitivity in a way that it has now reached a
physical limit. Significant further improvements with respect to sensitivity do not
seem possible in the near future (MicroCal, personal communication). Another
potential approach to decrease sample size may be miniaturization. Integrated
circuit microcalorimeters are being built and may allow the application of ITC to
micro-titre plate formats in the future.
Modern screening processes for drug candidates would profit from development
of high-throughput instruments. This would be a major breakthrough in the
detection of compounds in pharmaceutical chemistry. The current throughput only
allows measuring a limited number of promising candidates from a series which
could lead to missing candidates with unusual and unexpected binding character-
istics worth being further investigated. New calorimetric methods using such
technology are now being developed, and with this new methodology the
ORDER REPRINTS
calorimetric approach will undoubtedly play a dominant role in high-throughput

screening processes in the future.
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Thermodynamics of Protein

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Thermodynamics of Protein

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JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION

Vol. 24, Nos. 1 & 2, pp. 152, 2004

Thermodynamics of ProteinLigand Interactions:

Remo Perozzo,* Gerd Folkers, and Leonardo Scapozza

Department of Chemistry and Applied BioSciences, Swiss Federal

The understanding of molecular recognition processes of small ligands and

*Correspondence: Remo Perozzo, Department of Chemistry and Applied BioSciences, Swiss

DOI: 10.1081/RRS-120037896 1079-9893 (Print); 1532-4281 (Online)

2 Perozzo, Folkers, and Scapozza

in terms of thermodynamic parameters. Because heat changes occur in almost all

Key Words: Isothermal titration calorimetry; Proteinligand interaction;

A fundamental principle of all biological processes is molecular organization

Historical Background to Thermodynamics and Calorimetry

The background of the development of calorimetry and thermodynamics has

4 Perozzo, Folkers, and Scapozza

Despite this interesting experimental work, the resulting interpretations of the

every process, be it physical, chemical, or biological, is accompanied by heat

affordable commercial calorimeters, that made calorimetry to become an almost

CALORIMETRIC PRINCIPLES AND PROPERTIES

To avoid confusion in describing the principles of calorimetry and calorimeters

Calorimeters are instruments for quantification of heat effects. Several principles

6 Perozzo, Folkers, and Scapozza

is proportional to the heat quantity released in the experiment:

Operating Mode and Construction Design of the Instrument

thermal insulation from the surrounding thermostated bath, which forms

ISOTHERMAL TITRATION CALORIMETRY

The main calorimetric techniques applied to investigate biological macromole-

stoichiometry (n) of the association event. Moreover, experiments performed at

A number of suppliers offer microcalorimetric instruments with sufficient

introduction of the commercially available Omega titration calorimeter in 1989 (19),

8 Perozzo, Folkers, and Scapozza

General Experimental Setup

The setup of an ITC experiment is largely dependent on the thermodynamic

binding (107108 M1) requires low concentrations of the macromolecule. With

minor fraction of ligand is bound to the macromolecule, making it difficult to detect

10 Perozzo, Folkers, and Scapozza

To obtain high quality data, an appropriate protocol has to be established by

averaging the small heats at the end of the titration.

Evaluation of Protonation Effects

Whenever binding is coupled to changes in the protonation state of the system,

12 Perozzo, Folkers, and Scapozza

Ligand Binding in Titration Calorimetry

There are many techniques available to measure binding constants (KB).

The observable response of an ITC experiment is the heat change associated

Single Set of Independent Sites Model

14 Perozzo, Folkers, and Scapozza

Combining Eqs. (13) and (14) gives the quadratic equation

whose only meaningful root is

The integral heat of reaction Q after the ith injection is given by

Q nMT V0 Hi 17

qi nMT V0 H i i1 18

Complex Binding Models

BASIC THERMODYNAMIC RELATIONSHIPS

The binding enthalpy of proteinligand interactions can be determined

The Gibbs free energy is temperature dependent and is described by

where Cp is the heat capacity change and T0 is an appropriate reference

ST ST0 Cp lnT=T0 24

STRATEGIES FOR MEASURING

High-affinity binding constants for proteinligand interactions are inherently

16 Perozzo, Folkers, and Scapozza

possible to determine the binding constant for a proteinligand interaction under

Linked Protonation Effects in Ligand Binding

Figure 4. Scheme for proton binding linked to binding of a ligand L to a macromolecule M.

Thermodynamic Linkage to Temperature

where TS is the temperature at which there is no solvent contribution to the