Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242

www.elsevier.com/locate/pnmrs

NMR methods for the determination of protein–ligand

dissociation constants
Lee Fielding *

Organon BioSciences, Newhouse, Scotland ML1 5SH, United Kingdom

Received 27 February 2007

Available online 24 April 2007

Keywords: NMR spectroscopy; Protein-ligand interactions; Binding affinity; Quantitative methods; Drug discovery

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
1.1. Ligand–protein interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
1.2. NMR of dynamic equilibrium states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
1.3. Accuracy and precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
2. Evolution of methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3. Protein observed chemical shift titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
3.1. 1D 1H NMR studies of hevein domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
3.2. 1D 1H NMR studies of kringle fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
3.3. Other examples of 1H detected measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
3.4. Heteronuclear HSQC detected titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
4. Ligand observed methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
4.1. Ligand observed chemical shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
4.2. Ligand observed relaxation rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
4.2.1. Ligand observed transverse relaxation rates 1/T2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
4.2.2. Ligand observed longitudinal relaxation rates (1/T1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
4.3. Ligand observed translational diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
4.4. Ligand observed magnetization transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
4.4.1. Saturation transfer difference NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
4.4.2. WaterLOGSY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
4.4.3. Comment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
4.5. Ligand observed binding to serum albumins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
5. Competition binding experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
5.1. Graphical data treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
5.2. Magnetization transfer for sub micromolar binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
19
6. F NMR studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
6.1. Fluorine labelled proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
6.2. Fluorine labelled ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
6.3. Fluorine observed competition binding experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
7. KD from ligand dissociation kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

*
Tel.: +44 1698 736182; fax: +44 1698 736187.
E-mail address: l.fielding@organon.co.uk

0079-6565/$ - see front matter  2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.pnmrs.2007.04.001
220 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242

8. Alternative measures of protein–ligand binding affinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

8.1. Affinity index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
8.2. Affinity ranking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
8.3. NMR as a functional biological screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
9. Applications of CP-MAS NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

1. Introduction An earlier review [4] covered all of the early literature

1.1. Ligand–protein interactions
Molecular recognition lies at the heart of biological pro-
cesses. At the molecular level the biological activity of drugs
corresponds to the binding of small molecules to macromo-
lecular receptors, usually proteins. This binding process is
regarded as an equilibrium condition which results from a
balance between association and dissociation events. The
quantitative foundations of pharmacology are mathematical
models that describe these processes [1], and accordingly,
biological activity is expressed as the affinity of the partners
for each other as a thermodynamic equilibrium quantity.
Binding affinities are usually determined in a binding assay,
but increasingly in recent years, a large variety of physico-
chemical methods have been established for the quantitative
determination of ligand–protein dissociation constants.
This article looks at cases where NMR spectroscopy has
been used to determine the equilibrium binding constant
for small molecule–biomolecule complexes, with a focus
on ligand–protein work. There are many diverse strategies
for making these measurements by NMR, so the main aim
of the review is to discuss the available NMR experiments
and to illustrate, by means of examples the data analysis
methods that are applied. The variety of approaches to
analysis of NMR data can obfuscate the underlying sim-
plicity of the methods. It should be noted that identical
experimental approaches and data analysis methods are
used in other areas of chemistry, most notably in host–
guest chemistry. And, since such studies have been of inter-
est since the earliest days of NMR in chemistry, much of
what follows is not necessarily new.
An enormous amount has been published on NMR inves-
tigations of protein–ligand interactions. As much again has
been published on NMR studies of more general intermolec-
ular interactions involving species other than proteins.
Much of this literature deals with qualitative structural
issues. Useful earlier protein–ligand reviews that did address
quantitative issues are the works of Dwek [2] and Gemmec-
ker [3]. In order to have a clear focus and keep this review to
a manageable size, the following discussion deals only with
the quantitative analysis of protein–ligand interactions.
The discussion does not aim to completely cover everything
that has been published on quantitative descriptions of pro-
tein–ligand interactions, but it does aim to be wide ranging
and it does comprehensively cover all possible approaches.
pertaining to the determination of stability constants by
NMR methods. This earlier report describes the graphical
methods, which are now becoming only of historical inter-
ests. Examples were drawn from the field of host–guest
chemistry, but much is relevant to the study of the stability
of protein–ligand complexes. The monograph by Connors is
the definitive work on binding constants [5]. A binding
model is a prerequisite to any kind of data analysis. Usually
a 1:1 complex is assumed and this is very often taken for
granted. The classical NMR approach to the determination
of stoichiometry is the method of continuous variations
(Job’s method) [6], but this is rarely applied to protein com-
plexes. In most reports the stoichiometry of the complex is
either well known from other studies, or it is safely assumed.
An advantage of using NMR to measure protein–ligand
interaction is that the NMR method extends the range of
measurable interactions into the mM range, a region not
well covered by traditional biochemical binding assays.
1.2. NMR of dynamic equilibrium states
A protein and a ligand in thermodynamic equilibrium is
characterised by the dissociation constant KD, which for
the simplest case of a protein with a single binding site is
defined as
K D ¼ ½P½L=½PL; ð1Þ
where [P], [L] and [PL] are the equilibrium concentrations
of protein, ligand and complexed state, respectively. [P]
and [L] are also referred to as the free or non-bound states,
e.g., ‘free ligand’, ‘non-bound protein’; and [PL] is vary-
ingly referred to as ‘bound ligand’ or ‘saturated receptor’
depending on the context of the experiment. KD has the
units of concentration. Therefore a value of KD in the
mM range implies an approximately 1:1000 ratio of free
to bound states in an equimolar mixture of P and L and
a KD in the lM range implies an approximately
1:10,00,000 ratio of these states, i.e., a much more stable
complex with less of the ‘free’ species present.
In order to measure the dissociation constant of a pro-
tein–ligand complex we have to measure the equilibrium
concentrations of free and bound species. Traditionally this
is done by equilibrium dialysis, but a great variety of other
experimental approaches have been applied, including
NMR methods. To measure KD by means of NMR experi-
ments implies quantitative analysis of solutions that are
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
potentially lM in the observed nucleus. The object of the
NMR observation might be the ligand, or the protein or
both. In practice just one species is chosen as the observa-
tional target – the ligand or the protein and only very rarely
are both species viewed together. Hence, when viewing the
ligand, the NMR experiment has to be able to distinguish
between the free non-bound ligand and the ligand in the
bound state, so that [L] and [PL] can be quantified. Alterna-
tively, when observing the protein, the experiment has to dis-
tinguish between and quantify [P] and [PL]. Protein–ligand
complexes are dynamic systems and therefore the rate at
which the components of the protein–ligand complex
exchange between free and bound forms is central to the
NMR method.
For a system in slow exchange on the chemical shift time
scale (in general terms this would be a protein–ligand system
with high binding affinity and low dissociation constant,
KD = lM or lower), resolved signals might be expected for
the free and bound states, and using the ligand observed
example, [L] and [PL] are in principle available by integra-
tion of separate resolved signals. In practice this is extremely
difficult because of the difficulty of detecting lM signals in
what is likely to be a complex and crowded spectrum.
For a system in fast exchange the observed NMR
response of a ligand is the mole fraction weighted average
of the NMR parameters of the free and bound states
M obs ¼ X LðfreeÞ M LðfreeÞ þ X LðboundÞ M LðboundÞ ;
where Mobs is any NMR observable characteristic of the
equilibrium system, XL(free) and XL(bound) are the mole frac-
tions of free and bound ligand, and ML(free) and ML(bound)
are the NMR parameters of the ligand in its free and bound
states, respectively. Similarly for observation of the protein,
M obs ¼ X PðfreeÞ M PðfreeÞ þ X PðboundÞ M PðboundÞ ;
where XP and MP are now the mole fraction and the NMR
characteristic of the non-bound protein and occupied
protein. The mole fractions are defined as, XL(free) +
XL(bound) = 1 and XP(free) + XP(bound) = 1.
Describing species distributions in terms of mole frac-
tions is a very useful concept and ‘mole fraction bound’
is a term that occurs frequently in accounts of binding
interactions. It is generally used to describe the proportion
of a species present in the bound state relative to the total
amount. Both the ligand and the protein speciation can be
described in this way. So, whereas the mole fraction of
bound protein, XP(bound) might range from 0 to approach-
ing 1 over the course of a titration with a ligand, the mole
fraction of bound ligand XL(bound) might at the same time
range from near 1 to approaching 0. Although these terms
never reach their absolute limits (except for XP(bound) = 0 at
the start of a titration), in practice at experimental end
points they approach them closely enough that setting
X(bound) = 0 or 1 is a reasonable approximation. The
almost complete approach of one of these parameters to
the end point conditions (0 or 1) under conditions of satu-
221
1.20
1.00
Mole fraction bound
0.80
0.60
0.40
0.20
0.00
-0.20
0 20 40 60 80 100 120
[L] 0/[P]0
Fig. 1. Simulated speciation curves for protein (n) and for ligand (¤) for a
series of solutions with [P]0 = 0.1 mM, and [L]0 is incremented through
0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5–10 mM. The assumed association
constant for a single binding site is 2 mM. The solution composition is
presented as the ligand:protein ratio. Mole fraction bound represents the
concentration of species in the complexed state relative to the total
concentration of that species, XP(bound) and XL(bound) in the text.
rated binding sites is what allows graphical solutions to the
binding equations.
Fig. 1 illustrates the formation of a 1:1 protein–ligand
complex for a hypothetical low affinity system with
KD = 2 mM. The protein and ligand concentrations are
presented as a ratio, as is commonly encountered. This is
a typical example of NMR titration data. The XP(bound)
trace would result from observation of the protein NMR
ð2Þ
signals, and the XL(bound) trace would result from observa-
tion of the ligand. The figure shows that the protein speci-
ation varies considerably over the course of the titration,
and suggests that a spectroscopic detector that was sensi-
tive to this speciation would be a good means to measure
KD. The ligand speciation curve appears rather flat and
unpromising as a measure of KD, but under appropriate
ð3Þ conditions, ligand observations are just as effective.
The relationships between the relative responses of
XP(bound) and XL(bound) become clearer when the solution
composition is plotted on a log scale and this is shown in
Fig. 2A. Moreover, the dependence of this speciation on
KD is highly informative and this is shown in Fig. 2B and
C. It can be seen that as KD decreases (corresponding to
tighter binding) the protein and ligand speciation curves
become more like each other and more like standard dose
response curves. XP(bound) always starts at zero and may
closely approach the limit of 1 providing that KD is small
or that the titration reaches high enough ligand concentra-
tions to saturate the binding site. The starting value of
XL(bound) is very dependent on the simulated conditions
and in most practical cases will not even come close to
the limit of 1. XL(bound) will be approximately zero for
the duration of most NMR experiments. The usefulness
of the mole fraction units is that this is what is measured
by the equilibrium state analytical probe (the NMR data).
So, the process of measuring KD is one of transforming
mole fraction to concentration units (molarity).
Eqs. (2) and (3) are general for all NMR experiment
observables. The NMR parameter may be the position of
222 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242

1.2 where [L]0 and [P]0 are the total concentrations of protein
1.0 and ligand, respectively. These total concentrations are usu-
Mole fraction bound

ally known. They represent the composition of the solution

0.8
as constructed by the investigator. The key to the determi-
0.6 nation of KD then, is to relate the known solution composi-
0.4 tion [L]0 and [P]0 to the equilibrium concentrations of ligand
0.2 and protein, [L] and [P], by means of the NMR observables.
Note though that the relationship between Mobs and KD
0.0
is non linear, and the parameter ML(bound) or MP(bound) is
-0.2
frequently not observable, so that it cannot be directly
-2 -1 0 1 2 3
log [L]0/[P]0 determined. There are two ways forward. Traditionally,
experiment protocols have been designed that linearize
1.2 the relationship between Mobs and KD. The data are then
1.0 analysed graphically and Mbound and KD come from the
Mole fraction bound

0.8
slopes and intercepts. Alternatively (and more often), the
relationship between Mobs and KD is solved computation-
0.6
ally and data analysis consists of determining a computed
0.4 best fit binding curve to the experimental data [7–9]. This
0.2 involves a two parameter fit to a binding curve. The bot-
0.0
tom line is that it is not possible to measure KD from a sin-
gle NMR data point and NMR protocols invariably
-0.2
-2 -1 0 1 2 3
involve monitoring some NMR signal as a function of
log [L]0/[P]0 varying solution composition. It is useful to talk not about
absolute peak positions or linewidths, but the changes to
1.2 these parameters that occur during a titration. Hence:
1.0
Dobs ¼ M obs  M free ; ð6Þ
Mole fraction bound

0.8
is the change in the NMR parameter induced by the equi-
0.6
librium condition relative to the free or non-bound state,
0.4 e.g., the induced chemical shift of a protein proton caused
0.2 by adding an interacting ligand, and:
0.0 Dmax ¼ M bound  M free ; ð7Þ
-0.2
-2 -1 0 1 2 3
is the limiting case of the above expression. Following the
log [L]0/[P]0 above example, for a protein reporter proton this would be
the chemical shift difference between the bound and non-
Fig. 2. Simulated speciation plots utilizing a logarithmic concentration bound forms. These two general terms, Dobs – the observed
scale and showing the effects of varying KD. (a) The same data as for
effect at any arbitrary equilibrium state, and Dmax – the lim-
Fig. 1, utilizing log [L]0/[P]0, KD = 2 mM. (b) and (c), Speciation plots
simulated for identical solution compositions with, (b) KD = 0.2 mM, and iting effect representing a fully bound state occur repeat-
(c) KD = 0.02 mM. edly throughout the following text.

1.3. Accuracy and precision

a resonance expressed in ppm or Hz, a linewidth, or either
of the relaxation rates 1/T1 or 1/T2, or any other feature of
The highest quality information on KD is in the curved
the system that reports on the dynamic averaging, e.g.,
regions of graphs such as Fig. 1 and in the steepest gradient
NMR measured translational diffusion coefficients. The sit-
region of Fig. 2. The regions of slowly changing mole frac-
uation of fast exchange also corresponds to the condition
tion (near to 0 and 1) are regions where the analytical
of the great majority of NMR based KD measurements,
response is insensitive to solution composition. Several
and so forms of Eqs. (2) and (3), written in terms of a spe-
detailed analyses of the errors associated with fitting spectro-
cific NMR parameter account for a large part of what
scopic data to binding models have appeared [10–14]. The
follows.
issues of concern revolve around obtaining spectroscopic
For the single site protein (1:1 complex), the solution
titration data that is actually responsive to the equilibrium
composition is defined as:
state and is not simply a response to binding; and how much
½P0 ¼ ½P þ ½LP; ð4Þ of a binding isotherm needs to be observed in order to sup-
and port the hypothesis of the binding model. The difference
between observing an equilibrium state and observing a
½L0 ¼ ½L þ ½LP; ð5Þ binding event is illustrated by many of the figures in the liter-
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
ature. Straight line regions of binding curves correspond to
either the progressive saturation of a species that is present
in sub stoichiometric amounts, or to a titration end point
where one species is fully saturated. Such regions contain
no information on KD. Information on the equilibrium con-
dition is only present in the curved middle region of the
graph. The principal findings are as follows:
A ‘probability of binding’ (p) is defined as the ratio of con-
centration of complex to the maximum possible concentra-
tion of complex. This formulation recognises that the
maximum possible concentration of complex is always the
initial concentration of the minor component (usually pro-
tein). A ‘saturation fraction’ has also been defined as the
ratio between the actual complex concentration and the ini-
tial concentration of the reagent, the chemical shift of which
is being measured. This term is less useful for describing pro-
tein binding situations because it does not reflect the fact
that, at the start of the binding curve the concentration of
complex is limited by the concentration of added ligand.
The minimum error in the measurement of KD occurs at
p = 0.5, and the ‘best’ data are obtained from the range
0.2 < p < 0.8. In other words, the most accurate values
for KD are obtained when the equilibrium concentration
of the complex is approximately the same as the free con-
centration of the most dilute component.
The maximum information on the system comes from
studying the widest possible range of p. At least 75% of
the saturation curve is required in order to show correspon-
dence between the equation of the model and the equation
fitting the data (i.e., to verify that the binding model is
based on the correct stoichiometry). In other words any
binding data will be fit by any model over a suitably short
range of p. If the experimental data are limited, higher
order complexes should be verified to be absent. Determi-
nation of the stoichiometry of a complex requires measure-
ments at p = 1 (i.e., at undetectable protein or ligand
concentrations). Since these conditions are the opposite
of those required for an accurate measure of KD, the two
experiments should be separated.
The above comments cover the most important consider-
ations regarding experimental procedures. Some further rec-
ommendations for the optimization of experimental
conditions for the determination of Ka, written in the context
of host–guest chemistry, but appropriate to this discussion
have been discussed [4]. These mostly relate to the graphical
methods. Wilcox has discussed these issues from the perspec-
tive of a more up-to-date NMR curve fitting context [15].
This then is the reason that sensitivity considerations
limit the ability of NMR protocols to directly measure
KD. Regardless of which NMR parameter is observed,
the experiment must be sensitive to analyte concentrations
that are approximately of the same magnitude as KD. In
other words, NMR techniques cannot directly measure a
value of KD smaller than the limit of detection of the exper-
iment. Typically this is around 10–100 lM for routine
cases. The limit may be overcome by using competition
experiments, as described in Section 5.
223
2. Evolution of methods
Approaches to the study of protein–ligand interactions,
and the measurement of binding affinities, have naturally
been limited by whatever at the time was state-of-the-art
technology.
The organization of this review broadly reflects the pro-
gression of the implementation of methods. At the start
there was little choice. Only one or two methods were avail-
able. In the 1970s, the only way that the stability constant
of a protein–ligand complex could be measured by NMR
was by means of 1/T2 or 1/T1 relaxation rate observations.
With the available 100 MHz spectrometers, there was little
hope of resolving any protein signal. So the earliest NMR
measurements of protein–ligand stability constants were
based on relaxation rates of ligands in fast exchange with
the protein. Increases in spectrometer operating frequen-
cies coupled with the availability of pure protein and more
importantly, isotope enriched (15N and 13C) preparations,
allowed direct protein observed titrations starting from
the mid 1980s to the present. Now, twenty years later there
are many different tools available. Most recently, due to the
development of a new range of ligand observed methods
based on magnetization transfer effects, there has been a
return to ligand observed experimentation, with a lot of
interest in saturation transfer difference NMR.
Methods to deconvolute the concentrations of free and
bound species from exchange averaged NMR observations
will be presented in Section 3 and early in Section 4. This
represents the bulk of the published literature. The meth-
ods are conceptually easy to understand and easy to carry
out. Magnetization transfer difference experiments, which
effectively deconvolute the bound ligand fraction from
the total ligand concentration within the exchange experi-
ment, are the most up-to-date approaches and are consid-
ered at the end of Section 4. Most of the material in
Sections 5 and 6 can then be viewed as special cases and
as extensions of the general methods because no new
NMR detection is introduced. Some new ideas that work
best with 19F NMR are introduced at the end of Section
6. The review ends with a consideration of several other
diverse approaches that do not fit comfortably in the dis-
cussion so far.
NMR observations of protein–ligand systems are always
set up to observe either the protein or the ligand, never
both together. So it is convenient to organize the following
discussion according to whether the protein or the ligand is
the observed NMR active species. One of the challenges in
understanding the published literature in this area, is that
of understanding the various data treatments that have
been applied. As discussed by Conners [5], there are three
different ways in which to linearize the hyperbolic binding
curve. To add further confusion, sometimes the protein,
sometimes the ligand is at fixed concentration. Sometimes
neither component is held constant and the data analysis
applied may even be inappropriate. Add to this a few
unique and individual approaches to data analysis and this
224 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
leads to many representations of graphs that illustrate the
derivation of KD from NMR titration data.
3. Protein observed chemical shift titrations
In this class of experiments, an NMR property of a
nucleus in the protein is the marker of the binding equilib-
rium. Normally the response of this signal, perhaps the
chemical shift or linewidth of a protein proton, would be
monitored as ligand was titrated into the solution. It is
obvious that this method is only applicable to pure, solu-
ble, modest molecular weight proteins and is subject to
all of the constraints that go with protein structure investi-
gations by NMR. Isotope labelling is helpful, but not
essential. Titrating ligand into protein so that the ligand
eventually finishes in excess, thus saturating the protein
binding site is the only way to perform this study. Little
useful information would come out of a protocol where
the ligand concentration never exceeded that of the protein.
Neither will much useful information come from a system
where the ligand concentration vastly exceeds the protein
concentration unless the binding event is very weak (and
this case seems not to be interesting). Accordingly, the
experiments discussed in this section all follow protocols
where the ligand:protein ratio is varied within a modest
range, 0.1–3 or 5. The linear graphical methods that occur
widely in host–guest chemistry (see Box 1), are seldom if
ever applied to the study of protein–ligand interactions.
Box 1
There are three non-logarithmic linear solutions
to the NMR binding isotherm [5]. Written in terms
of the NMR observables (for a titration of ligand
into protein, and where the protein is the focus
of the NMR observation), they are
½L0 =Dobs ¼ ½L0 =Dmax þ K D =Dmax ;
Dobs =½L0 ¼ Dobs =K D þ Dmax =K D ;
1=Dobs ¼ K D =Dmax ½L0 þ 1=Dmax : ð10Þ
The graphs of these solutions are often referred
to by the names of early researchers, so that the
graph of Eq. (8) is a Scott plot, the graph of Eq. (9)
is a Scatchard plot, and that of Eq. (10) is the Bene-
si–Hildebrand plot. Connors terms them as a y-
reciprocal plot, an x-reciprocal plot and a double
reciprocal plot, respectively, according to whether
the dependent parameter (y the NMR observa-
tion), the independent parameter (x the ligand
concentration), or both appear in reciprocal terms.
This terminology has the advantage that it identi-
fies the plot more precisely, and avoids the confu-
sion caused by application of yet more names, as
occurs when parallel developments are made in
different fields, but communication between
different specialists does not take place. However
the aforementioned names have gained wide-
spread acceptance.
In the y-reciprocal plot (Scott plot) the ligand
concentration is plotted linearly along the
abscissa, thus retaining the scaling of the direct
binding curve, but straightening the line. The
NMR description of the bound state is obtained
from the reciprocal of the slope of the curve. The
y intercept (crossing the ordinate at [L]0 = 0) gives
KD/Dmax.
In the x-reciprocal plot (Scatchard plot) the
ratio of the NMR effect to ligand concentration is
plotted against the size of the NMR effect. The
slope of the line is the negative reciprocal of KD
and the NMR property of the bound state is
obtained from the intercept with the abscissa.
This data analysis has two distinct advantages
over the others, KD is obtained from the slope
of the curve, independent of any extrapolations,
and the graph is ‘closed’ so that extrapolation
at each end leads to an intersection with an axis.
The double reciprocal plot (Benesi–Hildebrand
plot) graphs the reciprocal of the NMR effect ver-
sus the reciprocal of the ligand concentration.
The slope gives KD/Dmax, and the intercept on the
y axis (1/[L]0 = 0), is the bound NMR parameter
(1/Dmax). This data treatment has the advantage
that it does not mix the dependent variable with
the independent variable (Dobs remains distinct
from [L]0).
Note that the protein concentration does not
appear in any of these solutions. So there is no
need for [P]0 to be accurately measured. The only
requirement is that [L]0 > [P]0. All three data treat-
ments are likely to be found in the literature, but
ð8Þ with the Scatchard and Benesi–Hildebrand
ð9Þ names occurring more frequently than Scott.
The Scatchard plot is generally preferred
because KD comes without extrapolation to a
potentially remote axis intersection.
The examples that follow are all based on the 1:1 bind-
ing model, and an assumption that the system is in fast
exchange. The case of insufficiently rapid exchange
between the two species being observed in the titration
(protein and bound protein) has been examined by Sudme-
ier et al. [16].
An advantage of protein observed NMR titrations, and
something that sets this method apart from ligand observed
NMR titrations, is that it is often possible to directly observe
the fully bound state. That is, a protein spectrum may be
obtained under conditions of a fully saturated binding site.
When this is the case, Dmax (corresponding to dbound) can
be measured directly, and at the risk of introducing some
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
greater variance in the result, one unknown drops out of the
binding isotherm. Most workers prefer to treat dbound as an
unknown and find the best fit of both parameters to a binding
curve, but KD is occasionally reported derived from the
(shorter) single parameter observation, see below.
3.1. 1D 1H NMR studies of hevein domains
The group of Jimenéz-Barbero has made extensive use of
protein observed chemical shift changes to quantify the bind-
ing affinity of carbohydrates to hevein domains [17]. For the
most part these studies exemplify the use of non-linear least
squares fitting of the observed 1H chemical shifts versus [L]0
binding curve to find values for KD and dbound.
Hevein is a small, cystein rich, single chain protein of 43
amino acids. The signals from Gln20 NH and Hc in the
1D 500 MHz 1H NMR spectrum can easily be followed dur-
ing titrations with chitobiose and chitotriose [18], or a variety
of N-acetyl glucosamine containing oligosaccharides [19], in
order to determine the association constants of the carbohy-
drate–protein complexes. In a typical experiment [P]0 was
around 0.7–1 mM and [L]0 reached 15 or 30 mM at the end
of the titration. The experiment protocol was designed so
that [P]0 remains constant as [L]0 varies. This simplifies the
data analysis. A further series of communications from this
group describe the thermodynamics of binding as deter-
mined by variable temperature 1H NMR studies and isother-
mal titration calorimetry [20–22]. The close agreement
between the NMR derived values and the directly measured
data is a verification of the applicability of the equilibrium
NMR method to systems with mM binding affinity.
3.2. 1D 1H NMR studies of kringle fragments
A sequence of papers from the group of Llinás exem-
plify the 1D 1H NMR chemical shift titration method
and illustrate a special data analysis for the case where
Dmax can be observed directly, thus in principle reducing
the dimensionality of the problem to one. But there was
some uncertainty in measuring the protein concentration
so [P]0 was treated as an unknown, thus increasing the
dimensions of the problem back to two.
Human plasminogen is a single chain protein of 791
amino acids. The molecule has a mosaic structure which
includes five kringle modules, each containing between 78
and 80 amino acids, and with a value of Mr of 9 kDa.
The binding of small molecule ligands to several of the
domains was studied by following the 1H NMR (300 or
500 MHz) chemical shifts of hydrogens on aromatic resi-
dues such as His, Trp and Tyr during a titration of the
ligand up to ratios of around 15:1. The values of KD were
extracted from the data by either a graphical method [23–
27], or by least squares fitting of the hyperbolic binding
curve [28]. In their (unique) graphical method the fraction
of ligand bound protein is obtained from the 1H NMR fre-
quency of the reporter proton in free protein (dfree), the
225
fully saturated state (dbound), and at the equilibrium condi-
tion (dobs) according to:
X PðboundÞ ¼ ½PL=½P0 ¼ ðdobs  dfree Þ=ðdbound  dfree Þ: ð11Þ
Assuming single site binding and fast exchange, it can be
shown that:
1=X PðboundÞ ¼ 1 þ K D f1=ð½L0  X PðboundÞ ½P0 Þg: ð12Þ
Thus a plot of 1/XP(bound) versus ([L]0 – XP(bound)[P]0) is linear
with slope KD and intercept 1. Since [P]0 was not accurately
known, it was considered an adjustable parameter and was
found by iteration to minimize the linear correlation coeffi-
cient of the plot. This is not a conventional data treatment
and does not correspond with any of the treatments in Box
1. The aromatic signals were selected for study because they
could often be detected as resolved signals in these modest
molecular weight proteins, and they were also sensitive to
the binding event. This later observation is indicative that
these residues are in the vicinity of the ligand binding pocket.
The 1H chemical shift changes induced by ligand binding are
small. Typically, they are within the range 0.01–0.1 ppm and
mostly only around 0.02–0.05 ppm. The values of KD were
found to be in the range 5 lM to 1 mM.
3.3. Other examples of 1H detected measurements
Trp–Trp (WW) domains are compact modules of 38–40
amino acids, folded into a three stranded b sheet. They are
found in single or tandem repeats in over 25 unrelated cell
signalling proteins. The binding of two phosphothreonine
peptides to a synthetic construct of the N-terminal WW
domain of Pin 1 has been studied by 600 MHz 1H NMR
titrations [29]. Addition of increasing amounts of peptide
ligands caused chemical shift changes for several protons
in the WW domain. During the titration several proton res-
onances broadened until they disappeared and then reap-
peared at large excess of ligand, indicating slow
exchange, with the difference in chemical shift between
the free and bound forms being of the same order of mag-
nitude as the exchange rate. The Ser11 amide proton reso-
nance which moved gradually and was more evidently in
fast exchange throughout the titration was chosen as an
appropriate marker of the equilibrium conditions. Titra-
tions with 1 mM protein, taken to [L]0/[P]0 ratios of 4.5
and 11, established values of KD of 117 and 230 lM for
the peptides by fitting the data to the equation
Dobs ¼ 0:5Dmax f1 þ X þ K D =½P0  ½ð1 þ X þ K D =½P0 Þ2  4X1=2 g
ð13Þ
where X is the molar ratio of ligand to protein.
During a study of bepridil binding to cardiac troponin C
it was noticed that addition of the drug caused well defined
and specific changes of chemical shift and linewidth in the
1
H NMR spectrum (360 MHz) of the protein [30]. These
changes occur throughout the spectrum. The most evident
changes occur in the aromatic region, in the region corre-
226 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
sponding to the terminal methyl groups of methionine res-
idues, and in the aliphatic methyl region. The chemical
shifts of three of these peaks plotted against [L]0/[P]0 pro-
duced smooth curves which reached limiting values near
the 1:1 ratio. The binding affinity (20 lM) was then esti-
mated by the abbreviated method using the directly
observed saturated shift as a reference. The concentration
of bound protein can be calculated from the known total
protein concentration using the relationship
Dobs ¼ ½PLDmax =½P0 ; ð14Þ
and KD can be estimated from Eq. (15) which is based on
the assumption that the concentration of bound ligand
equals that of bound protein,
K D ¼ ð½P0  ½PLÞð½L0  ½PLÞ=½PL: ð15Þ
A study of the interaction of DMSO with the FKBP12 pro-
tein is a good example of the utility of NMR to quantify
weak binding events [31]. Proteins commonly co-crystallize
with small molecules and solvents, and crystalline FKBP12
was known to be associated with six molecules of DMSO.
The NMR study was based on the specific perturbation of
a few 1H NMR chemical shifts of the protein (0.3 mM)
during a titration with up to 1.5 M DMSO. The binding
isotherm (Fig. 3) clearly covers a large part of the bound
mole fraction range (see Section 1.3), and is well fitted by
the 1:1 isotherm for KD = 275 mM, a very weak interac-
tion. Using an existing 1H assignment and the known ter-
tiary structure of the protein, it was possible to identify
the single binding site. Note that the plot in Fig. 3 is a clas-
sic example of an NMR titration curve and is identical in
form to that of Fig. 1. Dd is directly proportional to
XP(bound) as [L]0 is proportional to [L]0/[P]0.
3.4. Heteronuclear HSQC detected titrations
In these experiments ligand binding is detected from per-
turbations to the inverse detected 15N-1H HSQC spectra of
Fig. 3. Chemical shift changes (Dd) for one of the CcH3 resonances of Val-
55 of FKB12 protein as a function of DMSO concentration (correspond-
ing to [L]0). The curve represents the best fit solution of the quadratic
equation that describes 1:1 complex formation. The curve corresponds to
KD = 275 mM and Ddmax = 0.35 ppm [31]. Reproduced with permission.
 1999 Kluwer Academic Publishers.
the target protein. Dissociation constants are then obtained
by monitoring the chemical shift changes of the backbone
amide as a function of ligand concentration. This method
has been widely adopted. It is easy to implement, 15N
labelled materials are often available, the enhanced disper-
sion of 2D spectroscopy allows ready identification of suit-
able reporter signals (often several of them) and the high
sensitivity of gradient selected HSQC experiments allow
rapid data acquisition. This last point is a critical issue. It
is not practical to follow a titration strategy if the NMR
detection experiment takes a long time.
A study of the binding of three potential ligand peptides
to the SH3 domain from the Src family tyrosine kinase Fyn
exemplifies the experiment. The uniformly 15N labelled pro-
tein comprised 69 residues. The peptide ligands comprised
14 residues. Multiple binding curves were determined by
titrating each peptide into separate samples of 15N SH3
and acquiring 15N-1H HSQC spectra at as many as 14 differ-
ent ligand:protein ratios from the range 1:10 to 4:1 [32].
Chemical shift changes in both 1H and 15N dimensions were
logged and fitted by non-linear regression analysis to
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðK D þ ½L0 þ ½P0 Þ  ðK D þ ½L0 þ ½P0 Þ2  ð4½P0 ½L0 Þ
Dobs ¼ Dmax :
2½P0
ð16Þ
The measured KD values are 3 mM, 50 and 300 lM.
The group of Fesik in particular has pioneered the use of
protein observed 15N-1H HSQC as a tool for drug discov-
ery (SAR by NMR [33]) and has used 15N-1H HSQC spec-
troscopy for high throughput determination of values of
KD [34–37]. Data are fitted to a single site binding model,
using a least squares fitting search to find the values of
KD and the chemical shift of the fully saturated protein
as described above. In this way the protein binding affini-
ties of collections of several dozen ligands at a time can
be quantified. Observations of protein 15N-1H HSQC spec-
tra in conjunction with ligand titrations is now an estab-
lished favourite method for determining KD by NMR,
but with variations on the data treatment [38,39].
Sometimes Dmax is assumed to be the Dobs at highest
[L]0. Ligand induced chemical shift changes in the
15
N–1H HSQC spectrum of a recombinant two-domain
fragment of barley lectin revealed well resolved, indepen-
dent responses from the two domains, allowing the simul-
taneous determination of the binding affinities of both
sites [40]. Assignment of the 500 MHz 15N–1H HSQC spec-
trum gave dfree for each residue. Chemical shift changes of
several residues were then monitored during titration of the
ligand and dbound was taken from the observations at the
maximum ligand concentration (Mr 9 kDa; [P]0 1 mM;
[L]0–12 mM). This was justified because almost no change
in chemical shifts was observed between the last and the
penultimate titration point, indicating a saturation of bind-
ing sites. These limiting values were then used to translate
the chemical shift data at each titration point into the frac-
tions of occupied protein binding sites (fB and fC) at each
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
ligand concentration. The concentration of free ligand in
the sample is given by
½L ¼ ½L0  ½PLB   ½PLC ;
and the concentration of protein that remains unbound at
each domain is
½PB  ¼ ½P0  ½PLB  and ½PD  ¼ ½P0  ½PLD :
The equilibrium constant characterizing ligand binding to
any one domain is then given by
(   fied. The method that has been most widely used is that
1  ½L0 1 based on the relationship
fB ¼  fC  1  
2 ½P0 K D ½P0
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 2
½L 1 ½L
 f C  1  0 
½P0 K D ½P0
where f C is the average of the individual values of fC for
each monitored residue at domain C.
A natural continuation from the 15N–1H HSQC protein
detection approach is to apply 13C-1H HSQC detection.
Hajduk et al. have studied ligand binding to isotopically
13
C(methyl) labelled domain 3A of human serum albumin
[41]. More than 1800 ligands were screened and binding
was detected by acquiring 13C-1H HSQC spectra
(500 MHz) on 50 lM protein solution in the presence and
absence of added compound. The compounds were
assigned a score based on the magnitude of the change in
the HSQC spectrum and the highest scoring compounds
were progressed to a more complete characterisation by
means of additional ligand titrations. In this way 232
ligands with binding affinities in the range 10 lM to
2.0 mM were measured.
4. Ligand observed methods
The drive to make NMR into a tool for drug discovery
has lead to a resurgence of interest in ligand detected inter-
action studies [42–45]. There has been particular interest in
the transfer NOE experiment and many new ideas have
been based on intermolecular magnetization transfer.
Although primarily designed to detect ligand binding, these
experiments can also provide information on KD.
In these experiments the observed species is the small
molecule and it is almost always titrated into the protein
until present in a considerable excess concentration. It is
convenient to consider the experiments as being of two
types. In one class, conventional NMR parameters such
as chemical shift, linewidths, relaxation rates which report
on the equilibrium condition of the solution are measured.
These data are then processed in a similar fashion to the
preceding section after making appropriate adjustments
to the data analysis to account for the switch of observed
species. The other class of experiments are based on obser-
vations of intermolecular transfer of magnetization, and
these have become very popular recently. A major advan-
tage of monitoring the small molecule is that there is no
227
upper limit to the size of the protein that can be studied.
There is also no need to isotopically enrich the protein.
Most of the ‘‘traditional’’ ligand observed NMR studies
ð17Þ
have been analysed by linear methods. This is appropriate
because the requirement to observe any high resolution
ligand signal, that it must be in fast exchange with the
bound form, and present in considerable excess to the pro-
ð18Þ
tein concentration, is the same as that required to apply lin-
earized data analysis. Hence [L]0  [P]0, [L] = [L]0 and the
linearized form of the binding equation is completely justi-
½L0 ¼ ½P0 Dmax =Dobs  K D ; ð20Þ
 ffi)
4 0
 f C ;ð19Þ
½P0 and in this case, a plot of [L]0 versus 1/Dobs gives KD as
the intercept on the [L]0 axis.
This is a y-reciprocal plot in Connors’ terms because the
dependent variable is plotted on the reciprocal scale. These
plots are most frequently presented with [L]0 on the ordi-
nate (y axis) and 1/Dobs on the abscissa (x axis), which is
unfortunate because this orientation obscures any relation-
ship with other graphical methods. It would be preferable if
such graphs were rotated and inverted, so that [L]0 is plot-
ted along the abscissa. An alternative form which is also
encountered (usually in the 1/T1 studies) has the protein
concentration factored into the y axis
½P0 =Dobs ¼ ð½L0 þ K D Þ=Dmax : ð21Þ
For this solution a plot of [P]0/Dobs versus [L]0 gives KD
when the line is extrapolated to the x axis crossing. This
form is preferred because the graph is shown the right
way round and the slope gives directly, the bound NMR
parameter. Whichever plot is chosen, and whichever axis
is chosen to be plotted on the abscissa, all plots give
KD at the intersection with the [L]0 axis, independent of
[P]0.
It is worth noting again that in order to apply this kind
of data analysis one of the species present has to be main-
tained at a constant concentration during the course of the
titration. Usually this is the species which is not observed in
the NMR spectrum (protein in this section), but this is not
always the case and there are examples of titrations of pro-
tein into constant concentration solutions of ligand. In this
‘inverse’ protocol, it is still a requirement that [L]0 at all
times greatly exceeds [P]0. This requirement to control solu-
tion compositions has some implications for the design of
the experiment and may (due to solubility, stability and
availability) be difficult to achieve.
Two effects might interfere with this kind of study: fail-
ure to meet the fast exchange condition, and non specific
binding. Feeney et al. have made a study of the effects of
intermediate exchange processes on NMR observed bind-
ing curves [46]. For a nucleus on a ligand undergoing fast
chemical exchange between two sites, the transverse relax-
ation rate (and hence linewidth) is given by
228 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
1=T 2;obs ¼ X PL =T 2;bound þ X L =T 2;free þ X PL X 2L sB 4p2 Dd2 ;
ð22Þ
where sB is the lifetime of the bound state (equivalent to
1/koff). The last term is the exchange contribution, which
reduces to zero in the case of very fast exchange. The com-
mon assumption (implied throughout this review) is that
the protein–ligand complex is in fast exchange on the
NMR time scale, so that whatever NMR parameter is ob-
served, it is proportional to the mole fraction weighted
average of the bound and free states. For many such com-
plexes the association rate constant will be diffusion lim-
ited, with a value in the range 107–108 M1 s1. If the
ligand binds tightly (KD < 0.1 lM) the slow exchange con-
dition usually applies and separate spectra are observed for
the free and bound ligand. For ligands with weaker affinity
(KD > 1 mM) the fast exchange usually applies and there
are no problems. Feeney et al. point out that in order to
facilitate the analysis, there is often a general assumption
of fast exchange, without any check that this is in fact
the case. Considerable errors (up to two orders of magni-
tude) in KD can arise by indiscriminate assumption of the
fast exchange condition.
In a situation where the ligand is present in considerable
excess over the receptor it is likely that after saturation of
the specific binding site, the ligand will bind at non specific
sites. Hence, when interpreting the spectra it should always
be borne in mind that the spectra represent an average
across all the states that the nuclei experience. It may not
be known a priori how many states there are, or what their
parameters may be. This is a common and recognised
problem in studies of serum albumin (discussed in depth
later), and studies of whole biological entities, e.g., mem-
brane bound receptors. The presence of additional binding
sites has to be acknowledged and built into the model, and
this makes the binding models increasing mathematically
complex. Klotz has provided a very useful account of the
quantitative analysis of this kind of sequential binding [47].
4.1. Ligand observed chemical shifts
One of the early reported applications of this method is
the study by Perkins et al. of the binding of monosaccha-
ride inhibitors to hen egg-white lysozyme [48]. They used
270 MHz 1H NMR to observe the binding induced chemi-
cal shift of the sugar signals. This paper has a useful four
page appendix which derives formulae that are useful for
the analysis of the NMR data from ligand-macromolecule
equilibria, and gives formalisms for a ligand binding in sit-
uations other than the simple two site model.
The binding of sialic acid derivatives to haemagglutinin
was studied by following perturbations to the 500 MHz 1H
chemical shift of the sialic acid resonances in the presence
of protein [49]. The major perturbation observed was to
the chemical shift of the ligand N-acetyl methyl resonance,
presumably due to the proximity of this methyl group to
the shielded region of an aromatic residue in the binding
site. Several similar ligands were studied at concentrations
around 0.5–12 mM, and in the presence of typically 20–
40 lM protein. The 1H chemical shift effects observed were
typically of the order of 0.02 ppm at the highest [P]0/[L]0
ratios and values of KD in the range 2–10 mM were accu-
rately measured for several dozen ligand–protein systems.
Data were analysed by the above mentioned linearization
routine. Under the conditions where the fraction of ligand
bound to protein is small, KD is given by Eq. (20) and a
plot of [L]0 versus 1/Dobs gives KD at the y intercept. The
result is shown in Fig. 4.
The N-acetyl methyl group exhibits an upfield shift in all
of the ligands that bind. The 1H NMR shift of the bound
ligand is available from the extrapolation of the graph
shown and the upfield shift for this proton on all of the
ligands is around 2 ppm. This is consistent with a model
where the methyl protons sit directly over the six mem-
bered ring of tryptophan 153.
A study of sialyloligosaccharides binding to wheat germ
agglutinin dimers used the constant [P]0 (0.1 mM), [L]0
titration (0–15 mM) method again, but extended the data
analysis to include consideration of the ligand linewidths
and also incorporated the Swift-Connick equations [50]
as an additional means to estimate the NMR parameters
(shift and linewidth) of the bound ligand [51].
The binding of L-tryptophan to the trp repressor pro-
vides an example of a system where the ligand is not in fast
exchange between the free and bound states. The trp
Fig. 4. Ligand N-acetyl chemical shift data that were used to calculate KD
for interactions of sialyllactose isomers with haemagglutinins. Each plot
shows the variation of ligand Dd with [L]0 at a constant [P]0. Data for four
different systems are shown. Experiments in panels (a), (b) and (d) were
repeated at different protein concentrations, which is reflected in the fact
that the second plots have different slopes [49]. Reproduced with
permission.  1989 American Chemical Society.
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
repressor binds two molecules of tryptophan in two inde-
pendent sites with identical affinities. Since no cooperativ-
ity is involved, the system was treated as a simple two
site exchange model. In variable temperature experiments
at different protein:ligand ratios the L-tryptophan protons
were observed to be in the slow exchange, intermediate
exchange and fast exchange regimes. The low temperature
data were used to find dfree and dbound for the H-4 proton of
L-tryptophan. Full line shape analysis of ligand resonances
yielded the dissociation and association rate constants, the
binding constant, and the thermodynamic parameters of
the process [52].
4.2. Ligand observed relaxation rates
Because binding induced chemical shift changes are rel-
atively small compared to the linewidth changes, most
ligand observed NMR studies have focused on the relaxa-
tion rate effects. A review of binding induced relaxation
enhancements which includes an excellent discussion of
their application to the measurement of equilibrium bind-
ing constants is that by Ni [53].
4.2.1. Ligand observed transverse relaxation rates 1/T2
Fischer and Jardetzky performed the first quantitative
NMR estimation of a protein–ligand interaction with a
60 MHz 1H NMR study of the interaction of penicillin
binding to serum albumin, KD = 10–20 mM [54]. These
experiments were performed with penicillin solutions
around 10–500 mM and at a [L]0/[P]0 ratio of around
200:1. At 60 MHz, there was no prospect of making any
kind of useful observation of the protein. They estimated
that the maximum likely effect on the ligand chemical
shift upon binding in the vicinity of the diamagnetic
region of an unsaturated ring may be around 200 Hz
(0.33 ppm). Since only about 1% of the ligand was
expected to be bound, the observed change Ddmax for a
fast exchange system was predicted to be about 2 Hz
and this would not be detectable. However the ligand
linewidths were extremely sensitive to the addition of
albumin. This pioneering study demonstrated that the
penicillin–albumin system is indeed in fast exchange,
and that both KD and 1/T2bound could readily be extracted
from the titration data. The calculated relaxation rates (1/
T2bound) for bound penicillin were found to be in the
range 2000–7000 Hz. Shortly afterwards Gerig estimated
the 1H linewidths of tryptophan bound to a-chymotrypsin
at around 30–70 Hz, and put KD in the range 3–12 mM
by observing the ligand line broadening during the course
of a titration [55].
An instructive example is the one by Miller et al. [56]
which describes the binding of choline to the intact mem-
brane bound acetylcholine receptor by measuring the line-
width of the choline methyl 1H NMR (100 MHz) signal
during a titration of the ligand. An equilibrium dissocia-
tion constant of 190 ± 65 lM was obtained from five data
points (solutions were 5 lM in receptor and ranged from
229
0.23 to 1.1 mM in ligand). In another good example, the
low affinity interaction of antibiotics with bacterial ribo-
somes were quantified by following ligand 1H transverse
relaxation times, T2 measured by the Carr–Purcell–Mei-
boom-Gill spin echo method [57]. Ligand concentrations
were around 0.5–3 mM, ribosomes around 0.2–0.8 lM,
and KD values in the range 0.3–13 mM. The data analysis
is illustrated in Fig. 5.
Further discussion of the graphical fitting of linewidth
data can be found in a report on the binding of sialyloligo-
saccharides to wheat germ agglutin, itself a popular subject
of NMR protein–ligand studies [58]. The hits from an
exploratory screening exercise to find new inhibitors of
human factor Xa were followed by construction of 1H
NMR linewidth isotherms to establish quantitative binding
affinities [59].
4.2.2. Ligand observed longitudinal relaxation rates (1/T1)
It has been shown that the selective 1H longitudinal
relaxation rate (1/T1(sel)) of the ligand is a more sensitive
indicator of binding than is the nonselective rate [60].
Studies of agonist binding to the acetylcholine receptor
provide straightforward and clear examples of the use of
T1 data to measure dissociation constants. The relaxation
times were measured by the inversion recovery pulse
sequence and the data were analysed by means of the y-
reciprocal plot (Eq.(8)). In a ligand–receptor system where
the ligand is present in large excess over the receptor bind-
ing sites, the ligand relaxation rate is described by
1=T 1obs ¼ 1=T 1free þ fbound =ðT 1bound þ sbound Þ; ð23Þ
Fig. 5. (a) Generic data treatment for the graphical determination of KD
from ligand observed NMR data. KD is obtained from the vertical
intercept. The fast exchange parameters Mobs can be Dm, Dd, 1/T2 or 1/T1.
(b) Experimental data from the interaction of the antibiotic telithromycin
(ligand) with bacterial ribosomes. The plots shows [P]0/Dobs versus [L]0 for
five different reporter protons of the telithromycin molecule [57]. Repro-
duced with permission.  2000 The Royal Society of Chemistry.
230 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242

where fbound is the fraction bound ([PL]/[L]0) and sbound is be measured in NMR experiment times that are somewhat
the lifetime of the bound state. The relationship between longer than to those required to measure relaxation rates.
total ligand concentration and T1 is then The reason of course that the diffusion coefficient is use-
ful is that D is closely related to size. A small molecule will
½P0 T 1obs ¼ ð½L0 þ K D ÞðT 1bound þ sbound Þ; ð24Þ diffuse faster than a large molecule. So if the diffusion coef-
ficient of a small molecule is measured in the presence of a
and a plot of [P]0T1obs versus [L]0 gives a straight line with
large molecule to which it binds, the observed diffusion
KD obtained from the intercept of the x axis. This
coefficient will be the weighted average of the coefficients
approach was used to study the binding of various agonists
of the free and bound states. In the presence of a receptor
to the intact acetylcholine receptor of Torpedo californica
protein, the diffusion coefficient of the ligand will be less
Fig. 6, [61]. In an extension of this work, the binding of
than that measured for the free ligand. The usual assump-
a number of cholinergic agonists and antagonists to syn-
tions of fast exchange apply, only this time the ligand needs
thetic and recombinant peptides representative of subunits
to be in fast exchange on both the chemical shift time scale
of the receptor were studied [62]. In a typical experiment,
and also the timescale of the diffusion measurement (usu-
the ligand was titrated into a 0.2–0.3 mM solution of pro-
ally several hundred milliseconds). From the point of view
tein until ligand concentrations around 10 mM were
of data analysis, D is just another NMR parameter, like d
reached. It is not clear that the protein concentration was
or 1/T2 and (Eq. (2)) applies again. Hence,
properly held constant during this protocol.
The serum albumin system also provides several exam- Dobs ¼ X LðfreeÞ DLðfreeÞ þ X LðboundÞ DLðboundÞ : ð25Þ
ples of the use of ligand 1/T1 data to determine KD.
Because of the large amount published on this protein dis- Also, as in the earlier discussion, NMR diffusion studies of
cussion of serum albumins is deferred until Section 4.5. protein ligand interactions are just a special case of more
general intramolecular interaction studies, and again a
large amount of relevant information exists but without
4.3. Ligand observed translational diffusion
the ‘protein–ligand’ label. Two authoritative reviews that
cover the specific protein–ligand area are those by Price
NMR measurements of translational diffusion (D) by
[63] and Lucas and Larive [64].
means of pulsed-field gradient (PFG) spectroscopy have
Larive et al. have coined the term ‘diffusion dynamic
received much attention since the mid 1990s. Essentially
range’ to describe the ability of a PFG diffusion experiment
these experiments use spin echo pulse sequences with a z-
to respond sensitively to the binding constant, and there-
axis magnetic field gradient applied during the first dephas-
fore measure KD accurately [65]. In the context of the
ing time, and again after the refocusing pulse. The effect of
PFG experiment, KD is given by
the first gradient is to spatially encode and the second gra-
dient decodes the nuclear spins. Only spins that are still in K D ¼ ½P0 ðDbound  Dobs Þ=ðDobs  Dfree Þ
their original locations will contribute to the spin echo and
þ ½L0 ðDobs  Dbound Þ=ðDbound  Dfree Þ: ð26Þ
so it is possible, by incrementing the gradient strength or
duration, to measure molecular displacement or translation
diffusion coefficients. Many pulse programs have been
devised to optimize the performance and D can generally

Fig. 6. Plot of nicotine concentration versus [P]0 T1(sel) for multiple
measurements on two different acetylcholine receptor preparations (circles
and squares). The pyridinyl H-4 proton of nicotine was used for the
relaxation measurements. Similar results were obtained from the other
aromatic protons. The data show the typical scatter in the selective T1
measurements, and estimated error bars [61]. Reproduced with permis-
sion.  1988 Biophysical Society.
Fig. 7. Simulation of the diffusion dynamic range DD (DDfreeDDobs) as a
function of the ligand:protein ratio for various dissociation constants, (A)
KD = 1000 lM, (B) KD = 100 lM, (C) KD = 10 lM, (D) KD = 1 lM and
(E) KD = 0.001 lM. The simulation was performed for a fixed [P]0. The
diffusion coefficient of the ligand (DDfree) was set at 6.9 · 1010 m2 s1,
and that of the protein (DDbound) at 0.76 · 1010 m2 s1 [64]. Reproduced
with permission.  2004 Wiley Periodicals, Inc.
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
Fig. 7 was produced by calculating the values of DD for a
hypothetical system with ligand:protein molar ratios rang-
ing from 0.1 to 500 and for five different values of KD. The
useful dynamic range is region 2 where Dobs depends on
both KD and the ligand:protein ratio. Region 1 corre-
sponds to low ligand:protein ratios, Dobs–Dbound and DD
converges to the difference between Dfree and Dbound. At
too high ligand:protein ratios (region 3), Dobs–Dfree and
DD converges to zero.
Note that this graph is simply a special case of the dis-
cussion of Section 1.3. It is axiomatic that an experiment
aimed at measuring KD has to be sensitive to the binding
event. The diffusion experiment is measuring a property
of the ligand which changes as a function of the ratio of
the free and bound forms.
A 162 MHz 31P NMR study of the binding of 2,3-biphos-
phoglycerate to haemoglobin is the first documented appli-
cation of the diffusion technique to a protein–ligand
complex [66]. The diffusion coefficient of the ligand was mea-
sured from a series of solutions that were around 3–5 mM
haemoglobin and ranging from 2 to 80 mM ligand over 10
data points (see Fig. 8). The data analysis consisted of fitting
Dfree and KD to the titration curve, with Dbound set to the
value of Dprotein that was measured in a separate experiment.
The resulting values of KD for the binding of 2,3-biphospho-
glycerate with carbonmonoxy-, oxy-, and deoxy- haemoglo-
bin are, respectively, 1.98 ± 0.26 mM, 1.8 ± 0.5 mM and
Fig. 8. The diffusion coefficient of 2,3-biphosphoglycerate (DPG) in
carbonmonoxyhaemoglobin solutions as a function of [DPG]. Each value
of D was derived from a PFGLED NMR experiment. The three
parameters that define the solid line are the diffusion coefficient of non-
bound DPG (Dfree = 1.8 · 1010 m2 s1), the diffusion coefficient of the
carbonmonoxyhaemoglobin (Dbound = 0.1 · 1010 m2 s1), and the disso-
ciation constant (KD = 1.98 mM) [66]. Reproduced with permission. 
1994 Biophysical Society.
231
0.39 ± 0.26 mM. This plot shows how the observed diffusion
coefficient of the ligand is strongly attenuated by binding and
is very sensitive to the ligand:protein ratio at low [L]0
(<25 mM). At higher [L]0 most of the ligand is unbound,
so the curve levels and approaches Dfree.
Lennon et al. [66] noted that this PFG 31P NMR
method has an advantage over the more obvious 31P chem-
ical shift perturbation method because several factors other
than binding to haemoglobin influence the 31P chemical
shifts. The PFG NMR method is one of only a few tech-
niques available in which these interactions can be studied
in intact erythrocytes. However the experiments do take a
long time. Each titration data point is the result of an incre-
mented NMR experiment that especially at low ligand con-
centrations required substantial signal averaging. The data
shown in Fig. 8 are clearly in the correct part (region 2) of
Larive’s diffusion dynamic range graph. An earlier study of
the same 2,3-biphosphoglycerate/haemoglobin system adds
a novelty footnote to this discussion [67]. The 1990 study
was an equilibrium dialysis determination of KD and 13P
NMR was used to determine the concentration of 2,3-
diphosphoglycerate with the dialysis cell nestled inside a
10 mm NMR tube.
Some 15N filtered diffusion experiments have been used
to study of the binding of a peptide to the Src homology
3 domain of phox47 (60–70 amino acids). A binding iso-
therm was constructed from the diffusion coefficients of
the species in solutions that were 0.46 mM in protein and
ranging from 0.23 to 10.5 mM in ligand (ligand:protein
ratios from 0.5:1 to 23:1) and Dobs varied over the fairly
narrow range 0.95 · 1010 m2 s1 to 1.55 · 1010 m2 s1.
This was sufficient to measure KD at 21 ± 14 lM, compara-
ble with a value of 29 ± 3 lM established by fluorescence
spectrophotometry [68].
It is fair to ask ‘‘what is the advantage of diffusion based
estimates of KD over more classical relaxation rate experi-
ments?’’ The preferred experiment will be the one that best
discriminates between the bound and non-bound forms of
the ligand. For a small molecule binding to a large protein
in dilute non viscous solution D is likely to change by
around a factor of 10. This is a good ‘dynamic range’.
The relaxation rate difference between bound and free
states will be much more variable, depending on what
nucleus is observed and what relaxation rate is measured,
but will often be in the same range or greater. Probably
relaxation rate experiments are more direct and faster.
One clear advantage of the diffusion coefficient
approach is the possibility of determining KD from single
data point experiments, thus eliminating the need for
assembling a titration curve at different ligand concentra-
tions. In contrast to most of the preceding experiments
where the NMR parameter of the bound state (Dmax) can-
not be directly observed, (the exception is that sometimes
Dmax is assumed to have been observed in some protein
detected studies), the two limiting diffusion parameters,
that of the ligand (Dfree) and that of the protein–ligand
complex (Dbound) can both be directly measured in separate
232 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
PFG experiments. The diffusion coefficient of the bound
ligand is simply that of the protein. In practise many work-
ers continue to create binding isotherms from titration data
as discussed above, but the short cut route to single point
measurements is attractive for high throughput determina-
tions and has been demonstrated as a viable means to
quantitatively rank order screening hits from the SHAPES
strategy for drug discovery [69]. The cost of a faster exper-
iment is some degradation in the precision of the method.
Small errors in the measured diffusion coefficients arising
from any sources will result in large errors in KD. A simple
analysis based on propagation of random errors found that
in the specific case of the SHAPES screen a 5% error in
Dobs translated to a 125% error for KD = 1 mM, a 57%
error for KD = 100 lM, and a 127% error for KD = 10 lM
[69]. This is yet another manifestation of the material dis-
cussed in Section 1.3.
4.4. Ligand observed magnetization transfer
Two completely new NMR methods for measuring dis-
sociation constants have been available since 1999-2000.
Both techniques are based on observing the intensity of
magnetization transferred to free ligand via the bound
ligand from the protein protons. This is accomplished
either by direct selective excitation of the protein protons,
or by relaying magnetization via the solvent and protein.
The responses of magnetization transfer experiments are
not classical NMR parameters like chemical shifts or relax-
ation rates. They are exchange averaged parameters, but
their magnitudes are not simply determined by the respec-
tive populations and NMR parameters of free and bound
states, so Eq. (2) cannot be applied. The responses do
not have absolute values, they are dimensionless and are
affected by a host of experiment parameters. These experi-
ments are fundamentally different to those discussed in the
preceding sections because they report on the concentra-
tion of bound ligand [LP].
The well known transfer NOE effect might be regarded
as the forerunner to the following two methods. It is the
application of the 2D NOE experiment to exchanging sys-
tems. Typically a small molecule (ligand) is characterized
by a short correlation time and small positive NOE values.
A large molecule (protein) is characterised by a long correla-
tion time and large negative NOE values. So that for a pro-
tein–ligand system where the ligand is present in excess
[L]0 > [P]0 and is in fast chemical exchange, the large negative
NOE acquired by the ligand while it was at the binding site
overwhelms the small positive NOE of the unbound ligand.
The observed response will be dependent on the fraction of
bound ligand (amongst many other parameters) [70].
The potential of NOE experiments to be used quantita-
tively in this fashion seems not to have been explored
except to note that the affinity of ligands can be rank
ordered from the NOE pumping response [71]. A funda-
mental problem with using the transfer NOE response to
quantify binding is that it changes sign between the bound
and free states. Hence there is a risk that part of the bind-
ing curve will originate in a zero signal to noise region.
4.4.1. Saturation transfer difference NMR
The saturation transfer difference (STD) experiment was
devised to screen compound collections for binding activity
to proteins [72]. Saturation transfer refers to the mecha-
nism whereby magnetization introduced into a large pro-
tein molecule is able to very rapidly spread around all of
the constituent spins, including any attached ligand, such
that when the ligand leaves its binding site it carries with
it information in the form of spin polarization from the
protein. Thus the bulk NMR response of the ligand is an
averaged response, composed of the sum of signals from
non interacting ligands and previously bound ligands. This
saturation process is very efficient, so the modulation of the
ligand signal induced by the protein is readily detected,
even in the presence of a large excess of ligand. In order
to make the interaction apparent, the experiment is imple-
mented as a difference experiment. Two 1D spectra are
acquired– one with selective excitation of the protein
turned on, and one where the selective excitation is moved
to an empty spectral region. The difference spectrum will
show a response only of ligands that were at some time
associated with the protein. The sensitivity to discriminate
between binders and non binders is excellent, and this
experiment has been successfully used to identify ligands
with binding activity from multicomponent mixtures. The
STD experiment has been authoritatively reviewed by
Meyer and Peters [44]. A recent review by Krishnan con-
tains a good discussion of the quantitative analysis [73].
How is the STD response related to KD? Because the
STD experiment is a difference experiment, the STD spec-
trum contains only signals from the bound state of the
ligand. Hence the STD response reports on the concentra-
tion of the protein–ligand complex. As with all of the pro-
cedures so far, a titration with ligand is carried out to map
the response as a function of [L]0. Some normalization of
the STD signal intensity is then required. A relative STD
effect is defined by normalizing the STD signal to the inten-
sity of the same peak in the off-resonance spectrum, and
then a correction for total ligand concentration is intro-
duced to arrive at an ‘STD amplification factor’ ASTD. A
plot of the parameter ASTD versus [L]0 is a normal binding
isotherm and can be fitted to derive KD [74]. So, although
the observed STD NMR intensity is not a direct measure of
the affinity of a particular ligand, KD can be obtained from
the titration curve ASTD versus [L]0. The method is illus-
trated by Fig. 9 which shows the binding of methyl b-D-
galactoside to the 120 kDa lectin ricinus communis agglutin
I (RCS120) [74].
Ligand binding to human integrin aIIbb3 incorporated
into liposomes has also been studied in this way. This mem-
brane bound fibrinogen receptor consists of two sub units
125 and 108 kDa. The binding affinity of the peptide ligand
cyclo(RGDfV) was estimated at 30–60 lM, from observa-
tions of a solution that was 5 lM in protein and 29–
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242 233

[79]. The result is that the resonances of non-binding com-

pounds appear with opposite sign and tend to be weaker
than those of interacting ligands. In order to make quanti-
tative estimates of KD, some normalisation of the Water-
LOGSY signal intensity has to be made. This is necessary
because the ligand acquires some magnetisation directly
from bulk solvent irrespective of what is happening at the
binding interface. The correction is made simply by per-
forming the WaterLOGSY experiment again, this time on
the ligand solution without the protein present. The exper-
iment is performed at several different ligand concentra-
tions and the corrected response can be plotted against
[L]0 to produce the normal binding isotherm. This is anal-
ogous to the correction that has to be made to ASTD in the
previous experiment. The experiment is illustrated in
Fig. 10 with data from the tryptophan/human serum albu-
min system. The circles are the uncorrected responses and
Fig. 9. (a) The normalised saturation transfer difference response of the
the triangles are the response of ligand without protein that
H-4 signal of b-GalOMe as a function of [L]0/[P]0 during a titration of b-
GalOMe into 40 lM RCA120 protein. (b) Display of the same data in are used to apply the correction. The corrected response
terms of the STD amplification factor. This second plot shows that even has the profile of a familiar binding curve.
though the fraction of ligand which is saturated decreases at a higher
ligand concentrations, the absolute STD signal intensity increases in the
form of a saturation curve. The STD amplification factor is obtained by 4.4.3. Comment
multiplying the fractional STD effect shown in (a) with the ligand excess The magnetization transfer experiments are best viewed
[74]. Reproduced with permission.  2001 American Chemical Society. as a measure of the bound ligand concentration, for a sit-
uation where the proportionality constant between the
spectroscopic response and [PL] is unknown. In this respect
275 lM in the peptide, corresponding to ligand:protein
the data are not different to, for instance, the chemical shift
ratios from 6:1 to 55:1 [75]. A more recent STD NMR
titration where the limiting chemical shift is unknown. So
study reports on the binding of a peptidomimetic ligand
there is no difficulty in producing binding curves such as
to human CD4 protein [76]. STD amplification factor titra-
Figs. 9 and 10 and fitting them to a parabolic curve. KD
tion curves from several of the ligand protons were pre-
arises from the curvature, thus acquiring the concentration
sented. The STD derived KD (9 lM) compares favourably
with that derived from surface plasmon resonance (10 lM).
The STD method is greatly advantaged for ligand–pro-
tein interaction studies because it is a method that is com-
pletely unlimited by the size of the protein. It works better
for larger proteins. Most of the published STD NMR stud-
ies that have been published in the last five years are aimed
primarily at gaining structural information by epitope
mapping. These reports have not usually addressed the
issue of measuring dissociation constants, other than two
studies which rank-ordered the ligands according to their
(unquantified) binding affinities [77,78].

4.4.2. WaterLOGSY
In the second magnetization transfer experiment, bulk
water magnetization is transferred to the ligand via the
ligand–protein complex. This is termed WaterLOGSY
(Water-Ligand Observed by Gradient SpectroscopY).
The experiment relies upon the water molecules present
at the protein–ligand interface and uses intermolecular
NOE and chemical exchange with labile hydrogens to
transfer magnetisation from bulk water to the protein.
The acquired magnetization is in turn transferred to any
bound ligand which, when appropriate dissociation rates
apply, can leave the binding site carrying with it magnetisa-
tion which has the same sign as the starting magnetization
Fig. 10. WaterLOGSY responses for the C2-H resonance of L-tryptophan
as a function of ligand concentration during a titration into protein. The
circles and triangles are the experimental intensities recorded in the
presence and absence of 10 lM HSA, respectively. The WaterLOGSY
response without protein is linear with [L]0 and can be used to apply a
correction for effect of non bound ligand. The square points are the
intensity difference graph and the line is the best fit calculated quadratic
curve. The signal intensity is on an arbitrary scale [79]. Reproduced with
permission.  2001 Kluwer Academic Publishers.
234 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242

units of [L]0, and the proportionality constant is given by Table 1

the asymptote, but is a meaningless parameter. Dissociation constants (KD (mM)) and stoichiometries (n) obtained from
NMR studies of ligand binding to serum albumins
Neither the direct STD experiment, nor the direct
WaterLOGSY responses appear to have been widely used Protein Ligand NMR KD n Reference
method (mM)
for the determination of KD. The disadvantage of these
methods appears to be the need to correct the response HSA TFBAa D 2.2 9 [81]
BSA Phenylbutazone 1/T2 3 100e [82]
of these experiments with a second reference experiment BSA Azathioprine 1/T2 3 100e [83]
which doubles the length of what is already a lengthy pro- HSA Ibuprofen D 17 ± 4b 50 [84]
tocol. For a simple system consisting of only one protein HSA Ibuprofen 1/T1 49 ± 15b 36 ± 8b [84]
and one ligand, chemical shift or relaxation rate methods HSA Ibuprofen 1/T1 1.5 ± 0.2 22 ± 1c [85]
will be faster and easier to implement. The STD and HSA Ibuprofen 1/T1 2.0 ± 0.4 15 ± 2d [85]
BSA Ibuprofen 1/T2 50–100 3–7 [86]
WaterLOGSY experiments would be expected to work HSA Ibuprofen 1/T1 1.4 ± 0.2 33 ± 2 [87]
and would have an advantage when directly quantifying HSA Salicylate D 17 ± 8 35 [88]
binding in a mixture of tentative ligands. Both STD and HSA Salicylate 1/T1 15 ± 2 32 [88]
WaterLOGSY have been used to monitor binding (quanti- BSA Salicylate D 30 ± 4 33 ± 3 [89]
tatively) in competition binding studies and these studies BSA Salicylate Dd 1.2 ± 0.6 8±3 [86]
HSA Salicylate 1/T1 4.3 ± 0.5 35 ± 2 [87]
are discussed in Section 6.
a
4-Trifluoromethylbenzoic acid.
b
Entry is the simple mean and standard deviation of the range of results
4.5. Ligand observed binding to serum albumins quoted in the original communication.
c
pH 6.8.
Ligand detected studies of protein–ligand systems are d
pH 8.0.
e
vulnerable to interference from non-specific binding. Math- Assumed.
ematical models of multisite binding have been summa-
rized by Klotz [47]. The simplest approach, and the one
that is widely used, is to assume that the protein has n few) molecules that bind specifically make no significant
equivalent and non-interacting binding sites, contribution to the experimental observable which is dom-
inated by the behaviour of non-specific binding interac-
P þ nL ¡PLn
tions and free ligand.
and therefore The PFG diffusion method, relaxation rate methods and
n chemical shift perturbation methods have all been success-
K D ¼ ½P½L =½PLn : ð27Þ
fully applied in several studies of non-specific binding to
In this model the parameter n works as a scaling on the serum albumins. Table 1 summarises some of these studies.
NMR parameter of the bound state (Dmax). The bound This table illustrates the wide range of ligand observed
population of ligand given by NMR parameters that can successfully be used as a handle
1=2 on the dissociation constant. Also noteworthy are the high
X LðboundÞ ¼ a  ða2  bÞ ; ð28Þ
values of n showing that as well as being a promiscuous
where binder of ligands, serum albumins are also polygamous.
Also noteworthy are the wide range of reported values of
a ¼ ð½L0 þ n½P0 þ K D Þ=2½L0 ; ð29Þ
KD, indicating that either these data are not precise mea-
and sures, or that the system is itself is somewhat fuzzy. Both
explanations are valid. The discussion in the later salcy-
b ¼ n½P0 =½L0 : ð30Þ
late/BSA report contains a more complete account of
Eq. (28) can be inserted into Eq. (2) to calculate a binding non-specific drug-albumin interactions and NMR studies
curve to match experimental NMR data. The widespread [86].
use of this formula is undoubtedly due its simplicity rather It should be recognised that for systems with multiple
than its validity. It is straightforward to introduce the new binding sites, and where the stoichiometry (n) is introduced
term n into computer enabled data fitting routines. as an additional parameter, the binding curve is now a four
Serum albumins are able to bind drugs specifically at parameter fit (Mfree, Mbound, KD and n) and a cosmetically
high affinity binding sites (KD = lM), and non-specifically appealing fit is guaranteed to be obtained for the limited
at several, possibly dozens, of low affinity sites (KD = mM) number of data points that define the usual NMR experi-
[80]. It commonly occurs that different studies of drug- ment. Further, the four parameters are highly interdepen-
serum albumin interactions report binding affinities that dent and it may not be possible to obtain a unique
differ by orders of magnitude because different protocols solution. As always, a good fit of the calculated curve to
explore different parts of the specific–non-specific binding the experimental data is not a validation of the binding
continuum. Ligand observed NMR methods are carried model. A good fit of a four parameter curve to a handful
out at high ligand:protein ratios and so by definition are of data points should justly be viewed with some
weighted to report on non-specific binding. The one (or a scepticism.
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
5. Competition binding experiments
A general drawback of all ligand observed NMR meth-
ods is their failure to work directly with high affinity
ligands. It is generally accepted that the lower limit of
applicability of the NMR method is KD to 10–100 lM.
Ligands with dissociation constants lower than this are
tightly bound which means that there is little exchange with
free ligand during the time scale of the NMR experiment
(typically a few hundred milliseconds) and therefore no
information about the bound state is detectable. This limit
has become smaller as the sensitivity of NMR hardware
has increased. This difficulty can be overcome by exploiting
the well known phenomenon of competition binding. The
binding experiment is performed in the presence of a sec-
ond ligand which occupies the same binding site as the tar-
get ligand. The competition process effectively modulates
the NMR response of the observed ligand and scales it
back into the region p = 0.2–0.8. No new NMR detection
techniques are introduced in the following discussion.
The experiments are based on exactly the same parameters
(e.g., linewidths, relaxation rates, and NOE responses) that
have been previously described in Sections 3 and 4, except
that they are now applied to a three component system.
The experiment protocols and the data analysis methods
are the points of interest.
A few of the competition methods have been purpose
designed to push the limits of NMR quantification to
sub-micromolar levels. Other studies have arisen from
NMR screening methods to identify new ligands for phar-
macologically interesting receptors and were driven by a
need to circumvent the false negative issue with high affin-
ity ligands. Although designed primarily to detect and sig-
nal the binding event, it turns out that these data can also
be used, often quickly and with little extra effort, to quan-
tify KD.
5.1. Graphical data treatment
A study of the binding of some sialic acid derivatives to
haemagglutinin in intact influenza virus is the first reported
application of quantitative NMR competition methods to
the study of protein–ligand interactions [90]. Although
nowadays this method is not likely to be used, this commu-
nication is instructive because it contains clear accounts of
the data analysis. The linewidth of the N-acetyl or O-
methyl 1H resonances of a reporter or reference ligand
(L2) were followed during titration with sialic acid deriva-
tives of interest (L1). Typically titrations were carried out
at 1 mM in the reference ligand and the ligand of interest
was incremented up to 15 mM. As is usual in graphical
methods the solution compositions were contrived to bring
about a reduction in the number of degrees of freedom of
the system. In this case the titrations were performed at
constant reference ligand concentration [L2]0 and constant
protein concentration [P]0. Graphical data analyses were
presented for two possible cases depending on whether
235
Fig. 11. 500 MHz 1H NMR linewidth data from the titration of a(2,6)-
sialyllactose (L1) using Neu4Pp5Aca2Me (L2) as a reporter ligand to
probe the interaction with haemagglutinin. [L2]0 is fixed at 1.0 mM and
[P]0 is fixed at 2 mg/ml. (a) The linewidths of two protons (N-acetyl methyl
and 2-O-methyl) on the reporter ligand (L2) as a function of [L1]0. (b) The
response of the N-acetyl methyl protons of the analyte ligand (L1) as a
function of [L1]0. In both cases KD is derived from the y- intercept [90].
Reproduced with permission.  1992 Academic Press, Inc.
L1 or L2 is the observed species. In the case where the con-
centration of the measured ligand [L1]0 is varied while the
NMR property of the reference ligand L2 is observed
 
K D DmaxðL2Þ ½P0 ½L20
½L10 ¼  KD 1 þ ; ð31Þ
K DðL2Þ DobsðL2Þ K DðL2Þ
and a plot of [L1]0 versus 1/Dobs(L2) is a straight line with a
y-intercept of –KD(1 + [L2]0/KD(L2). Thus, if KD(L2) has
been measured independently, KD can be determined from
the chemical shift or (as in this case) from the linewidth
behaviour of species L2 as a function of [L1]0. This proto-
col is useful for ligands whose resonances are difficult to
analyse due to overlap or poor signal-to-noise ratio. The
outcome is illustrated in Fig. 11a from which KD =
2.7 ± 0.2 mM.
The other scenario is where the NMR parameter of the
titrating ligand L1 is monitored as a function of varying
[L1]0. In this case it can be shown that
 
DmaxðL1Þ ½P0 ½L20
½L10 ¼  KD 1 þ ; ð32Þ
DobsðL1Þ K DðL2Þ
and a plot of [L1]0 versus 1/Dobs(L1) is again a straight line
with a y-intercept of –KD(1 + [L2]0/KD(L2). This is illus-
trated for the same ligand system in Fig. 11b, which leads
to KD = 1.8 ± 0.5 mM.
In these experiments the reporter ligand has a binding
affinity which is not very different from those of the inter-
esting ligands. It is an important study because it expands
236 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
the applicability of the NMR titration method to systems
where tighter binding constants may be determined. Eqs.
(31) and (32) are based on an assumption that the concen-
tration of free ligand is approximately the same as [L]0, so
the dissociation constants that can be measured are limited
to the range [P]0.
5.2. Magnetization transfer for sub micromolar binding
Some more recent developments to competition binding
have successfully extended the range of NMR measurable
values of KD to lM levels. The interest in this topic can
be traced back to a communication from Mayer and Meyer
[74]. They observed that the STD response of a bound
ligand was reduced by the presence of a competitor ligand,
and noted that it is possible to determine the KD value of a
ligand from the IC50 value of any competitor ligand which
has a known dissociation constant, thus
K D ¼ ð½L10 K I Þ=ðIC50ðL2Þ  K I Þ:
In the above equation, and in the remainder of this section,
the symbol KI is used to indicate the dissociation constant
of the competitor, reference or reporter ligand (L2) and KD
is used to indicate the dissociation constant of the ligand of
interests (L1).
Dalvit [79,91] recognised that the waterLOGSY
response (see Section 4.4.2) of a system comprising of a
receptor and a specific, but medium affinity (mM) ligand
could be used as a screen to detect the presence of compet-
ing higher affinity ligands. Remembering that the water-
LOGSY response arises from bound ligand, any molecule
that competes with the bound ligand will result in a
decrease in intensity of the waterLOGSY signal. Knowl-
edge of the binding affinity of the reporter ligand, coupled
with some careful experiment design permits quantitative
determination of the binding constant for the competition
ligand. Setting up the experiment conditions (solution com-
positions) so that waterLOGSY responses of investigative
ligand (L1), reporter ligand (L2) and protein are compared
with directly corresponding reporter ligand (L2) and pro-
tein data simplifies the data analysis. The relationship
between attenuation of waterLOGSY response I(+)/I()
and the dissociation constants of the two ligands is given
by
  rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n 
½L20
I ðþÞ ½P0 þ ½L10 þ K D 1 þ KI  ½P0 þ ½L10 þ K D 1 þ K I 0
¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 2
I ðÞ
½P0 þ ½L10 þ K D  ½P0 þ ½L10 þ K D  4½P0 ½L10
Fig. 12 is plotted directly from solutions of Eq. (34) to illus-
trate the dependence of the response ratio I(+)/I() on [P]0,
[L1]0, [L2]0, KD and KI, respectively [91]. This chart demon-
strates that the ratio I(+)/I() is effectively modulated as a
function of the inhibitor binding affinity. The inhibitor dis-
places the monitored ligand, thus reducing the water-
LOGSY response. The chart shows that more effective
inhibitors (KI approaches zero) displace more of the ob-
Fig. 12. WaterLOGSY signal attenuation of the reference compound as a
ð33Þ function of the dissociation constant KI of the competitor. The simulation
was performed using Eq. (34) with a competitor concentration of 5 lM, a
reference (ligand of interest) concentration of 50 lM, and a protein
concentration of 2 lM [91]. Reproduced with permission.  2002 American
Chemical Society.
served ligand and the ratio I(+)/I() approaches zero. The
four curves illustrate the dependence of the function on
the binding affinity of the reference molecule. Weaker affin-
ity reporter ligands are more easily displaced. The effect of
higher affinity reference molecules is to move the curves to-
wards the left and top of the graph (mirroring the effect of
decreasing KD).
The experiment has been validated with the model sys-
tem serum albumin with 6-methyltryptophan as a reference
ligand (KD = 37 lM) and diazepam as the high affinity
competitor ligand. A single point experiment established
a 65% reduction in the reporter ligand waterLOGSY signal
when diazepam was added. This translated to
KD = 2 ± 1 lM for the diazepam binding [91]. The same
group have demonstrated that transverse relaxation and
longitudinal relaxation parameters of a reference ‘reporter’
ligand are equally effective for quantifying high affinity
binding [92]. Further work on competition methods from
this group are discussed in Section 6.3.
The HSA/tryptophan/diazepam system was also used
o2 recently to demonstrate that competition STD NMR can
½L2
 4½P0 ½L10 also be put into a quantitative context [93]. The principles
and experiment protocols are almost identical to that
ð34Þ
described above, except that magnetization transfer is
induced by the STD pulse sequence instead of water-
LOGSY. Again, binding of the high affinity ligand is sig-
nalled by a decrease in the intensity of the reporter ligand
STD response, and the magnitude of this attenuation is
used to deduce KI for the strong binder. The dissociation
constant of diazepam was estimated at 2.4 ± 0.5 lM from
the observed fractional reduction of 0.41 in the 6-methyl-
tryptophan STD signal. Fig. 13 is complementary to
Fig. 12. It shows the modulation of the STD response of
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
Fig. 13. STD signal reduction of the indicator as a function of the
competitor concentration. The concentration of the STD indicator
(KD = 10 lM) and the protein are 100 and 5 lM, respectively. Simulations
were performed according to Eq. (20) for five different dissociation
constants (KI) of the inhibitor (100, 10, 1, 0.1 and 0.01 lM). The STD
signal reduction is expressed as a fraction and is equal to the ratio of the
STD signal intensities in the presence and absence of the competitor. [P]0 is
included in the simulation. The dashed lines represent simulations with [P]0
omitted [93]. Reproduced with permission.  2004 John Wiley & Sons, Ltd.
a reporter ligand as a function of the inhibitor concentra-
tion for a range of values of KI. This chart shows that
the fractional reduction of the STD signal of the reporter
is most sensitive to the competitor concentration in the
range 0.2–0.8 (this is yet another statement of the material
in Section 1.3). The chart suggests that there is no lower
limit to the KI values that can be measured in this way.
The KI of any higher affinity ligand can be accurately mea-
sured by lowering the concentration of inhibitor until the
STD modulation is in the 0.2–0.8 range [93].
Another discussion of NMR detected competition bind-
ing noted that KI was in principle available from knowl-
edge of the KD of the reporter ligand but stopped short
of reporting quantitative figures [94].
19
6. F NMR studies
Introduction of a fluorine atom into either the protein or
the ligand opens the door to 19F NMR observation and
this has an enormous range of benefits. 19F has nearly
the same sensitivity as 1H. It is a spin 1/2 nucleus and its
chemical shift range in proteins and small organics is usu-
ally around 100 ppm. There is essentially no biological
background of 19F signals, thus only the site of interest will
appear in the experimental spectrum. These factors make
the 19F NMR spectrum easy to acquire and easy to analyse.
Furthermore, 19F occurs frequently as a motif in man made
drugs (it is usually put in to improve adverse metabolic
profiles). Consequently there is a large body of literature
that reports on the use of 19F NMR to study protein–
ligand interactions [95,96].
From the perspective of quantifying the protein–ligand
interaction, nothing that follows is new to what has been
presented in the preceding sections. The concern is still to
237
deconvolute the bound and free mole fractions of the spe-
cies that is observed and the only change is that we are now
looking at 19F as a reporter nucleus. This review could well
have been written with the discussion of 19F experiments
sprinkled around the text under the other appropriate
sub headings. However there is a didactic advantage in pre-
senting this material separately. At the risk of being repet-
itive, the papers cited in the following discussion illustrate
all of the experiment protocols and data analysis tech-
niques again, but often with greater clarity.
6.1. Fluorine labelled proteins
A study of oligosaccharide binding to AcAMP2-like
peptides provides a recent example. A 30 amino acid
residue analogue corresponding to the hevein domain was
synthesised with Phe18 and Tyr20 changed to 4-fluorophe-
nylalanine. The mM binding of chitotriose was evaluated
from 19F NMR titration data [97].
6.2. Fluorine labelled ligands
The binding of N-trifluoroacetyl-D-(and L-)p-fluorophe-
nylalanine to a-chymotrypsin was quantitatively measured
from perturbations to the 19F chemical shifts of both fluo-
rine nuclei upon interaction with the active site [98]. The
downfield shifts of the bound ligand are attributed to inter-
actions with His, Ser and Asp residues in the binding
pocket. The communication includes a full and clear
description of a unique data analysis which is based on
arithmetic iterations of the quadratic binding equation to
determine Dmax and KD. Although obsolete, the procedure
is worth noting. Mathematical procedures to account for
dimerization of the protein add interest to the data analy-
sis. Data were collected under conditions of constant [P]0
(1.9 mM) and varying [L]0 (from 0.5 to 20 mM); and for
the reverse case of constant [L]0 (1.5 mM) and varying
[P]0 (0.05 to 1.9 mM). It was found that the L isomer binds
about 10 times less strongly than the D isomer
(KD = 0.32 mM at pH 6.0).
A consequence of the large chemical shift range of 19F
is that dynamic systems may often be in the intermediate
or slow exchange regime when observed via 19F. In the
slow exchange case the data treatment becomes trivial.
Integration of the free and bound signals gives [L] and
[LP] directly. Much of the 19F NMR protein–ligand
interaction literature reports on systems that are on this
time scale. A study of the binding of trifluoromethyl ana-
logs of 6,7-dimethyl-8-ribityllumazine to lumazine apo-
protein is such an example. The bound ligands give
rise to additional broad signals several ppm downfield
of the free ligand signal, KD = 3–5 lM, analysed by Scat-
chard plots [99]. This system is discussed further in Sec-
tion 7.
19
F spin–spin relaxation rates (1/T2) measured by the
CPMG sequence have been successfully applied to measure
the low affinity binding of the volatile anaesthetic isoflu-
238 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
rane to BSA [100]. In this study, only one 19F signal was
observed, but exchange is sufficiently slow that exchange
broadening had to be allowed for [54]. This was achieved
by varying the interval between the 180 refocusing pulses.
The data analysis then proceeded exactly as described pre-
viously in Section 4.2. A plot of 1/T2(obs) versus [L]0 gives –
KD at the x intercept, (1.4 mM).
6.3. Fluorine observed competition binding experiments
The large chemical shift anisotropy of 19F results in very
broad lines for bound fluorinated ligands and therefore
very large differences in line widths between the bound
and free states. This makes fluorinated ligands particularly
well suited for competition binding experiments. The trans-
verse relaxation rate 1/T2 of fluorine in a reference ligand is
an ideal parameter to monitor as a function of test ligand
concentration. For instance, the low affinity ligand 2-
hydroxy-3-fluorobenzoic acid has been used as a reporter
ligand for the Sudlow site 1 of human serum albumin. Dis-
sociation constants for hundreds of compounds in the
range from a few nM to high lM are claimed to have been
measured by this method [101].
Dalvit et al. have recently provided an in depth theoret-
ical analysis of 19F NMR competition binding using weak
affinity reporter ligands. This includes several instructive
simulations showing the relationships between the equilib-
rium parameters – fraction bound, fractional reductions in
NMR responses etc., and the defining parameters of the
system – KD, KI, [P]0, [L1]0, and [L2]0 [102].
7. KD from ligand dissociation kinetics
KD was defined earlier (Eq. (1)) in terms of the equilib-
rium concentrations of bound and free species. An equiva-
lent definition of KD is in terms of the equilibrium
condition established by the balance of the ligand associa-
tion (kon) and dissociation (koff) rates
K D ¼ k on =k off : ð35Þ
So instead of quantifying the solution speciation, one
might aim instead at measuring these rates. In very general
terms the first order ligand dissociation rate (units M1) is
a reflection of the strength of the intermolecular complex,
whereas the ligand association rate is a measure of how
quickly the ligand can arrive at the protein. An assumption
is frequently made that the kon is diffusion limited and is
thus independent of the system. A value of 1 · 109 M1 s1
is usually cited for kon. When this condition is satisfied koff
is a useful proxy for the dissociation constant. There are
many NMR protocols for measuring these rates [103].
Information on ligand exchange kinetics (koff and kon
rates and hence KD) can be derived from complete line shape
analysis of individual NMR peaks. Jardetzky et al. have
reported on a 1H NMR (500 MHz) study of the binding of
L-tryptophan to the trp repressor of Escherichia Coli [52].
This study is interesting because it used a full line shape anal-
ysis of the ligand H-4 proton over a range of protein and
ligand concentration (mM) and over a temperature range
from 20 C where the system is in slow exchange to 65 C
when it is in fast exchange. This thorough analysis results
in a full thermodynamic picture of complex formation in
the form of a KD versus 1/T Arrhenius plot.
The thermodynamic and kinetic parameters associated
with the binding of N-acetylgalactosamine to Artocampus
integifolia agglutin were determined from the temperature
dependence of line broadening in the 19F and 13C NMR
spectra of the ligand [104]. In this system chemical shift
changes were not observed on binding. Campbell et al.
reported a full line shape analysis of the 1D profiles of indi-
vidual 15N–1H HSQC peaks at each point of a titration of a
12-residue phosphopeptide into a solution of the SH2-N
domain of the p85a subunit of PI 3 0 -kinase [105]. This anal-
ysis gave information about the kinetics of complex forma-
tion in a system with KD in the nM range. A software tool
is available to facilitate the analysis of line shapes from
titration generated two-dimensional spectra [106].
Again, 19F NMR observations of fluorinated ligands
provide some of the clearest examples of such studies. All
of the kinetic parameters, including kon and KD were
obtained from quantitative analysis of NOESY spectra of
the lumazine protein/ligand system [107]. Peng has
described in detail the use of cross-correlated 19F relaxation
measurements for the study of ligand–receptor interactions
and show how these data provide estimates of KD [108].
8. Alternative measures of protein–ligand binding affinity
Although the dissociation constant KD is the preferred
quantitative measure of stability for bimolecular complexes
(because its meaning is clear), some other measures of affin-
ity are also used. It may not always be possible to define the
binding interaction in terms of a simple 1:1 complex. Some-
times an experiment observable is related to KD, but in an
indeterminate way, so that the response can only be used as
an indication of binding strength or as a ranking parame-
ter, but not as an absolute measure.
8.1. Affinity index
Rossi et al. have advocated the ‘affinity index’ as a mea-
sure of ligand macrocycle affinity [109]. Using the selective
spin–lattice relaxation rate R1 (1/T1(sel)) as the NMR obser-
vable, it can be shown that
 
1 1 1
¼ þ ½L ; ð36Þ
DR KD R1ðboundÞ ½P0
and therefore a plot of D1/T1(sel) versus the protein concen-
tration will be a straight line passing through the origin and
with a slope
 
T K D R1ðboundÞ
½AL ¼ ; ð37Þ
1 þ K D ½L
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
which is defined as the ‘affinity index’. The dimensions of
T
½AL are M1 s1 and the T and L superscripts and sub-
scripts signify the temperature and ligand concentration
at which the measurement was made. The advantage of this
term is that it provides a measure of ligand-macromolecule
global affinity which is independent of the number of bind-
ing sites. A disadvantage of this parameter is its depen-
dence on the ligand concentration. So, although it can be
a useful way to rank order a series of ligands (or receptors)
within a single study where [L]0 can be controlled, it is
not a very efficient way to communicate knowledge of
receptor–ligand binding strength. The method has been
used to study the interaction of carbamazepine with
albumin [110], chloramphenicol and thiamphenicol with
albumin [111], and anandamide with multiple cannabinoid
receptors [112].
8.2. Affinity ranking
NMR is now commonly used in the pharmaceutical
industry as a lead discovery tool in the drug discovery pro-
cess. Relatively small compound collections are screened
for receptor binding and the NMR response is used to sig-
nal binding. The usual outcome from such an endeavour is
a subset of ‘hits’ that have some affinity for the receptor. It
is highly desirable to rank order these hits, but there may
not be enough time or resource to apply the titration meth-
ods. One crude but pragmatic way forward is to rank the
ligands according to the magnitude of the NMR measured
binding response.
A search for ligands of human adipocyte fatty acid bind-
ing protein (FABP4) is a recent example. A 1H 1D T1q
relaxation filter experiment was used to identify ligands
from a collection of 531 compound which was initially
screened as cocktails of 5–10 compounds. The ligands were
then classified as weak or strong binders according to the
amplitude of the attenuation response in a second T1q
relaxation filter experiment applied with a shorter spin lock
time [113].
8.3. NMR as a functional biological screen
The requirements of the pharmaceuticals industry to
invent more effective lead discovery programs has also
led to the development of an NMR based functional
screen. A functional screen is one where tentative new
drugs are tested against the fully functional target, e.g.,
an enzyme that is actively turning over a substrate. The
method, termed 3-FABS (three fluorine atoms for bio-
chemical screening) by its inventors, uses NMR to analyse
the progress of the enzyme reaction and is able to report
the 50% mean inhibition concentration (IC50) of the active
ligand [114,115]. It is interesting that neither the receptor
(the enzyme), nor the screened ligand are observed in this
experiment. The method requires the substrate of the
enzyme to be labelled with 19F. NMR analysis of the reac-
tion progress is then based on straightforward integration
239
Fig. 14. IC50 of an experimental drug (known as H89) determined by 19F
NMR detection of the CF3 labelled starting and enzymatically modified
substrate. The test system comprised of a protein kinase and a 19F labelled
substrate peptide. This plot shows the unphosphorylated peptide concen-
tration, [pep], as a function of the inhibitor concentration [H89]. The best fit
inhibition curve to the experimental data gives an IC50 of 0.72 ± 0.05 lM
[114]. Reproduced with permission.  2003 American Chemical Society.
of 1D 19F NMR data to quantify the amount of substrate
and the product of the enzyme reaction. The compound
library is added to 384 well plates containing the active
enzyme and substrate and the reaction is quenched at a
time when some fraction of the substrate is expected to
be consumed. Automated processing of the 19F NMR spec-
tra is able to quickly identify the active compounds. The
IC50 can be determined by collecting a full inhibition bind-
ing curve (Fig. 14), or it can be estimated approximately
from a single point measurement because the values for
both plateaus of Fig. 14 are known from references and
blanks.
Cryogenically cooled probes permit the application of
this kind of screening with femtomole levels of target
enzyme, a concentration similar to that required for tradi-
tional high throughput screening methods [102].
9. Applications of CP-MAS NMR
No information about the equilibrium binding affinity is
available from solid crystalline protein–ligand complexes.
However the techniques of solid state NMR can be usefully
applied to the hydrated gelatinous samples that are typical
of membrane bound protein preparations. Cross-polariza-
tion magic angle spinning (CP-MAS) NMR goes some
way to resolving the technical difficulties of studying very
large proteins embedded in a lipid membrane. Add to this
the simplification resulting from isotope labelled ligands,
usually labelled at a single site with either 13C, 15N or
19
F, and direct observation of the dynamics of receptor
bound ligands becomes possible. Unlike the high resolution
solution phase techniques which sample an exchange aver-
aged population, the CP-MAS experiment only sees the
ligand that is bound to the receptor. Thus the few reports
of quantitation of KD by these methods are a special case
of ligand observed experiments.
240 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
CP-MAS works because it distinguishes bound from
non-bound ligand. The 13C CP-MAS experiment generates
signals from 13C nuclei in rigid solids by transferring mag-
netization from abundant 1H nuclei. The experiment first
excites 1H magnetization and then transfers magnetization
from 1H to 13C during the contact time. The 1H-13C dipole
coupling is averaged to zero for rapidly reorienting small
molecules, so the 13C magnetization in the non-bound
ligand remains at equilibrium (weak), and is essentially
not detected. If at some point during the contact time
(spin-lock field applied) the ligand binds to the protein, it
will build up 13C magnetization at a predictable rate and
will produce a signal during the detection period. The
result is that the CP-MAS experiment can give a direct read
out of [PL] unperturbed by [L]. Measuring [PL] is the key
to knowing KD.
A study of the weak affinity binding of galactose to the
lactose transport protein LacS in native membranes is the
first reported estimation of KD from CP-MAS data [116].
Use of [1-13C]D-galactose allowed clear observation of the
bound ligand against the background of natural abun-
dance carbon. Cross polarization NMR had not been
thought to be amenable to quantitative interpretation since
the response of the observed nucleus is dependent on multi-
ple and difficult to quantify interactions with nearby spins.
However, a selectively bound substrate should experience a
consistent environment throughout a titration, and the
response can be analysed as a function of [L]0. This is
shown in Fig. 15. The two data points at the highest [L]0
were deemed to have been corrupted and were discarded
from the Scatchard plot. Note that the normalization step
in this example assumes that saturation binding has
occurred at 5 mM ligand (certainly not the case) and is a
drastic simplification of the data analysis.
Fig. 15. Binding isotherm for [1-13C]-D-galactose to LacS membranes as
determined from the intensity of the substrate signal in the CP MAS 13C
spectrum. The ratio of occupied binding sites was established by
normalizing the response to the maximum observed response (the response
at 5 mM ligand). The inset is a Scatchard plot of the first four data points
[116]. Reproduced with permission.  1999 American Chemical Society.
Fig. 16. Simulations of cross polarization intensity profiles for a ligand
interacting with a membrane protein, calculated for different values of the
dissociation rate constant (koff) and the dissociation constant (KD). The
virtual system was [L]0 = 6 mM, THC = 1.5 ms, T free 1qH ¼ 100 ms, and
T bound
1qH ¼ 2 ms. The simulations represent [P] 0 = 2.4 mM (dashed line),
2.0 mM (solid line) and 1.6 mM (dotted line) [117]. Reproduced with
permission.  2004 American Chemical Society.
The rate at which 13C magnetization builds up in the
ligand is a balance between the positive addition of magne-
tization through the dipolar coupling and loss through
relaxation. These factors in turn depend on the binding
constant and also the number of times that the ligand
might exchange on and off the protein during the contact
time. Hence the response is a function of both KD and koff.
Simulated profiles of expected signal intensity are shown in
Fig. 16. The experiment has been demonstrated with stud-
ies of the binding of methyl [1-13C]-b-D-glucuronide to the
GusB membrane transport protein from Escherichia coli
[117], and as a 19F NMR version for the anti-psychotic
drug trifluoroperazine binding to membrane embedded
gastric H+/K+ ATPase [118]. Under optimum conditions
the variable contact time CP-MAS experiment can be a sin-
gle point experiment. It is able to establish KD from a single
protein/ligand sample at just one protein:ligand ratio.
10. Conclusion
The use of NMR to determine quantitatively the associ-
ation constants of protein–ligand complexes is firmly estab-
lished. NMR might not always be the optimum means to
measure KD for a system, but NMR specialists obviously
enjoy pushing the boundaries and expanding the applica-
bility of magnetic resonance methods and the result is that
there are a lot of NMR experiments available for the task.
The tried and tested titration methods of sections 3 and 4
are used very widely.
The opportunity to observe cleanly the species of inter-
est with high sensitivity and often with no additional chem-
ical manipulation has resulted in the accumulation of huge
experience with 19F NMR. Few would consider putting
 L. Fielding / Progress in Nuclear Magnetic Resonance Spectroscopy 51 (2007) 219–242
fluorine into a ligand solely for the purpose of measuring
KD perhaps, but for systems where fluorine is already pres-
ent in the ligand, the 19F NMR experiments might be con-
sidered as first choice methods for measuring KD.
The most recent developments with magnetization
transfer experiments, competition binding and CP-MAS
approaches, have resulted in novel, sensitive and specific
NMR methods to measure KD that have moved far from
the original linewidth and chemical shift perturbation
approaches. They illustrate the breadth of interest in this
science and suggest that more interesting developments will
continue to come. The most useful experiments will be
those that establish [PL] directly and expeditiously.
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