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BIOCHEMISTRY I

Midterm exam schedule (Chap 2-7)


4/24 (Fri) 8pm
No withdrawal allowed!

Key term report submission:


3/4
3/9.
3/16.
3/23.
4/1.
4/8.

Chap 2. aqueous system.


Chap 3. amino acids, peptides, and proteins
Chap 4. tertiary and quaternary structure of proteins
Chap 5. protein functions
Chap 6. enzymes
Chap 7. carbohydrates and glycobiology

# Quizes
3/23 (Mon): Chapter 2 and 3
4/6 (Mon): Chapter 4 and 5

3 | Amino Acids, Peptides, Proteins

2013 W. H. Freeman and Company

CHAPTER 3
Amino Acids, Peptides,
Proteins
Learning goals:

Structure and naming of amino acids


Structure and properties of peptides
Ionization behavior of amino acids and peptides
Methods to characterize peptides and proteins

Proteins:
Main Agents of Biological Function
Catalysis
enolase (in the glycolytic pathway)
DNA polymerase (in DNA replication)

Transport
hemoglobin (transports O2 in the blood)
lactose permease (transports lactose across the cell membrane)

Structure
collagen (connective tissue)
keratin (hair, nails, feathers, horns)

Motion
myosin (muscle tissue)
actin (muscle tissue, cell motility)

Proteins serve a wide range of


biological functions

Amino Acids:
Building Blocks of Protein
Proteins are linear heteropolymers of -amino acids
Amino acids have properties that are well-suited to carry
out a variety of biological functions

Capacity to polymerize
Useful acid-base properties
Varied physical properties
Varied chemical functionality

Amino acids share many features,


differing only at the R substituent

Most -amino acids are chiral


The -carbon always has four substituents and is
tetrahedral
All (except proline) have:
an acidic carboxyl group
a basic amino group

an -hydrogen connected to the -carbon

The fourth substituent (R) is unique


In glycine, the fourth substituent is also hydrogen

All amino acids are chiral (except glycine)


Proteins only contain L amino acids

Absolute configuration: the configuration of four different


substituent groups around an asymmetric carbon atoms, in
relation to D-and L-glyceraldehyde
=> D, L system

Amino Acids: Atom Naming


Organic nomenclature: start from one end
Biochemical designation:
start from -carbon and go down the R-group

Amino Acids: Classification


Common amino acids can be placed in five basic
groups depending on their R substituents:
Nonpolar, aliphatic (7)
Aromatic (3)
Polar, uncharged (5)
Positively charged (3)
Negatively charged (2)

These amino acid side chains absorb UV light at 270280 nm

These amino acids side chains can form hydrogen bonds.


Cysteine can form disulfide bonds.

Aromatic R Groups
These amino acid side chains absorb UV light at
270-280 nm

FIGURE 3-6 Absorption of ultraviolet light by aromatic amino acids

Protein concentration measurements?

Measurement of light absorption by a spectrophotometers


used to detect and identify molecules and to measure their
concentration in solution

The fraction of the incident light absorbed by a solution


at a given wavelength is related to the thickness of the
absorbing layer (path length) and the concentration of
the absorbing species. These two relationships are combined into
the Lambert-Beer law
Io
log
= cl
I

=> absorbance, A

Io is the intensity of the incident light


I is the intensity of the transmitted light
I/Io (the inverse of the ratio in the equation) is the transmittance
is the molar extinction coefflcient (in units of liters per mole-centimeter)
c is the concentration of the absorbing species (in moles per liter)
l is the path length of the light-absorbing sample (in centimeters)

BOX 3-1 FIGURE 1 The principal components of a


spectrophotometer.

Absorbance A is directly proportional to the concentration


of the absorbing solute.

varies with the nature of the absorbing compound, the


solvent, and the wavelength, and also with pH if the lightabsorbing species is in equilibrium with an ionization state
that has different absorbance properties.

Cysteine can form disulfide bonds

FIGURE 3-7Reversible formation of a disulfide bond by the oxidation of two molecules of


cysteine. Disulfide bonds between Cys residues stabilize the structures of many proteins.

You should memorize

- One letter codes


- Three letter codes
- Chemical structure of 20 amino acids

Uncommon Amino Acids in Proteins


Not incorporated by ribosomes
except for Selenocysteine
Arise by post-translational modifications of
proteins
Reversible modifications, especially
phosphorylation, are important in regulation and
signaling

Modified Amino Acids Found in Proteins

Reversible Modifications of Amino Acids

Ionization of Amino Acids


At acidic pH, the carboxyl group is protonated and
the amino acid is in the cationic form.
At neutral pH, the carboxyl group is deprotonated
but the amino group is protonated. The net charge is
zero; such ions are called Zwitterions.
At alkaline pH, the amino group is neutral NH2 and
the amino acid is in the anionic form.

Zwitterion: a dipolar ion with spatially separated positive and


negative charges.

FIGURE 3-9 Nonionic and zwitterionic forms of amino acids.

FIGURE 3-10 Titration of an amino acid.


Cation Zwitterion Anion

Chemical Environment Affects pKa Values


-carboxy group is much more acidic than in carboxylic acids
-amino group is slightly less basic than in amines

Amino acids can act as buffers


Amino acids with uncharged side chains, such as glycine,
have two pKa values:
The pKa of the -carboxyl group is 2.34

The pKa of the -amino group is 9.6


It can act as a buffer in two pH regimes.

Buffer
Regions

Amino acids carry a net charge of zero


at a specific pH (the pI)
Zwitterions predominate at pH values between the pKa values of
the amino and carboxyl groups
For amino acids without ionizable side chains, the Isoelectric Point
(equivalence point, pI) is
pK1 pK 2
pI
2

At this point, the net charge is zero


AA does not migrate in electric field

Ionizable side chains can show up


in titration curves
Ionizable side chains can be also titrated
Titration curves are now more complex
pKa values are discernable if two pKa values are more

than two pH units apart

How to Calculate the pI When the


Side Chain is Ionizable
Identify species that carries a net zero charge
Identify pKa value that defines the acid strength of this
zwitterion: (pK2)
Identify pKa value that defines the base strength of this

zwitterion: (pK1)
Take the average of these two pKa values
What is the pI of histidine?

Formation of Peptides
Peptides are small condensation products of amino acids
They are small compared to proteins (Mw < 10 kDa)

Peptide ends are not the same


Numbering (and naming) starts from the amino terminus
AA1

AA2

AA3

AA4

AA5

Naming peptides:
start at the N-terminus
Using full amino acid names
Serylglycyltyrosylalanylleucine

Using the three-letter code abbreviation


Ser-Gly-Tyr-Ala-Leu

For longer peptides (like proteins) the oneletter code can be used
SGYAL

Sample problem

Draw the chemical structure of the following peptide at pH 7.

CHEMIST

p.111
11. Net Electric Charge of Peptides A peptide has the sequence

GluHisTrpSerGlyLeuArgProGly
(a)What is the net charge of the molecule at pH 3, 8, and 11? (Use
pKa values for side chains and terminal amino and carboxyl groups as
given in Table 31.)
(b) Estimate the pI for this peptide.

Peptides: A Variety of Functions


Hormones and pheromones
insulin (think sugar)
oxytocin (think childbirth)
sex-peptide (think fruit fly mating)

Neuropeptides
substance P (pain mediator)

Antibiotics
polymyxin B (for Gram bacteria)
bacitracin (for Gram + bacteria)

Protection, e.g., toxins


amanitin (mushrooms)
conotoxin (cone snails)
chlorotoxin (scorpions)

Proteins are:
Polypeptides (covalently linked -amino acids) + possibly:

cofactors

functional non-amino acid component


metal ions or organic molecules

coenzymes

organic cofactors
NAD+ in lactate dehydrogenase

prosthetic groups

covalently attached cofactors


heme in myoglobin

other modifications

Polypeptide size and number


varies greatly in proteins

Classes of Conjugated Proteins

What to Study about Peptides and


Proteins
What is its sequence and composition?
What is its three-dimensional structure?
How does it find its native fold?
How does it achieve its biochemical role?

How is its function regulated?


How does it interacts with other macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?

A mixture of proteins can be separated


Separation relies on differences in
physical and chemical properties

Charge
Size
Affinity for a ligand
Solubility
Hydrophobicity
Thermal stability

Chromatography is commonly used for


preparative separation

Protein separation
Crude extract
released proteins into a solution after breaking
open the cells

Separate proteins into different fractions


(old fashion) Protein solubility: pH,
temperature, salt concentration
(now) column chromatography

Column Chromatography

Separation by Charge

p.111
5. Separation of Amino Acids by Ion-Exchange Chromatography Mixtures of
amino acids can be analyzed by first separating the mixture into its components
through ion-exchange chromatography. Amino acids placed on a cation-exchange
resin (see Fig. 317a) containing sulfonate (-SO3- ) groups flow down the column
at different rates because of two factors that influence their movement: (1) ionic
attraction between the sulfonate residues on the column and positively charged
functional groups on the amino acids, and (2) hydrophobic interactions between
amino acid side chains and the strongly hydrophobic backbone of the polystyrene
resin. For each pair of amino acids listed, determine which will be eluted first from
an ion-exchange column by a pH 7.0 buffer.
(a) Asp and Lys
(b) Arg and Met
(c) Glu and Val
(d) Gly and Leu
(e) Ser and Ala

Separation by Size

Separation by Affinity

Modern chromatographic methods employing HPLC

High pressure pump, higher quality chromatographic materials


=> Reducing the transit time on the column, limit diffusional spreading

Electrophoresis for Protein Analysis


Separation in analytical scale is commonly
done by electrophoresis
Electric field pulls proteins according to their
charge
Gel matrix hinders mobility of proteins according
to their size and shape

SDS PAGE: Molecular Weight


SDS sodium dodecyl sulfate a detergent

SDS micelles bind to and unfold all the proteins


SDS gives all proteins an uniformly negative charge
The native shape of proteins does not matter
Rate of movement will only depend on size: small
proteins will move faster

SDS-PAGE can be used to calculate the


molecular weight of a protein

p. 111
10. Subunit Composition of a Protein A protein has a molecular
mass of 400 kDa when measured by gel filtration. When subjected
to gel electrophoresis in the presence of sodium dodecyl sulfate
(SDS), the protein gives three bands with molecular masses of 180,
160, and 60 kDa. When electrophoresis is carried out in the
presence of SDS and dithiothreitol, three bands are again formed,
this time with molecular masses of 160, 90, and 60 kDa. Determine
the subunit composition of the protein.
Answer The protein has four subunits, with molecular masses of
160, 90, 90, and 60 kDa. The
two 90 kDa subunits (possibly identical) are linked by one or
more disulfide bonds.

Isoelectric focusing can be used to


determine the pI of a protein

Isoelectric focusing and SDS-PAGE are


combined in 2D electrophoresis

Quantification of unseparated proteins

Activity: total units of enzymes in a solution


Specific activity: the number of enzyme units per milligram of
total protein

Specific activity (activity/total protein)


can be used to assess protein purity

Levels of structure in proteins

-Primary structure
: a description of all covalent bonding (peptide bonding, -S-S-)
: sequence of amino acid residues
-Secondary structure
: particular structural arrangements of amino acids giving rise to
recurring structural patterns
-Tertiary structure
: 3D folding of a polypeptide chain
- Quaternary structure
: arrangement in space of 2 or more polypeptide subunits

FIGURE 3-23 Levels of structure in proteins.

The function of a protein depends on its amino acid sequence

Each type of protein has a unique amino acid sequence.


Amino acid sequence determines 3D structure and ultimately
its function.
If primary structure alters, the function may also be
changed.
Functionally similar proteins from different species often
have similar amino acid sequence.
But, 20-30% of the proteins in humans are polymorphic.

The amino acid sequences of millions of proteins have been determined

Determination of amino acid sequence from polypeptide


chain is difficult
Amino acid sequence in protein is related to nucleotide
sequence
DNA sequence => decoding genetic code => protein amino
acid sequence

Protein Sequencing
It is essential to further biochemical analysis that we know
the sequence of the protein we are studying
Actual sequence generally determined from DNA sequence
Edman Degradation (Classical method)
Successive rounds of N-terminal modification, cleavage, and
identification
Can be used to identify protein with known sequence

Mass Spectrometry (Modern method)


MALDI MS and ESI MS can precisely identify the mass of a
peptide, and thus the amino acid sequence
Can be used to determine post-translational modifications

Edmans Degradation

FIGURE 3-27 Cleaving proteins and sequencing and ordering the


peptide fragments.

Amino acids sequences can also be deduced by other methods


Mass spectrometry

Analytes
Ionization in vacuum: generating charged analytes
apply them into an electric/magnetic field
paths through the field are a function of mass-to-charge ratio (m/z)
Matrix-assisted laser desorption/ionization mass spectrometry
(MALDI MS)
Light absorbing matrix: laser light
protein ionization and desorption into vacuum
Electrospray ionization mass spectrometry
(ESI MS)
Analytes pass through a charged needle kept at high electric potential
dispersing the solution into a find mist of charged microdroplets.

Electrospray mass spectrometry of a protein.

A protein solution is dispersed into highly charged droplets by passage


through a needle under the influence of a high-voltage electric field. The
droplets evaporate, and the ions (with added protons in this case) enter
the mass spectrometer for m/z measurement.

Electrospray mass spectrometry of a protein

The spectrum generated (b) is a family of peaks, with each successive


peak (from right to left) corresponding to a charged species increased by
1 in both mass and charge. Inset: a computer-generated transformation of
this spectrum.

Tandem MS (MS/MS)
To sequence short polypeptide

peptide separation by first MS


fragmentation by high-energy impact with collision gas
b-type ion: charge retained on N-terminal side
y-type ion: charge retained on C-terminal side
measure m/z ratio by second MS

BOX 3-2 FIGURE 2a Obtaining protein sequence information


with tandem MS.

Obtaining protein sequence information with tandem MS.

Protein Sequences as Clues to


Evolutionary Relationships
Sequences of homologous proteins from a wide range
of species can be aligned and analyzed for differences
Differences indicate evolutionary divergences
Analysis of multiple protein families can indicate
evolutionary relationships between organisms,
ultimately the history of life on Earth

Chapter 3: Summary
In this chapter, we learned about:

The many biological functions of peptides and proteins


The structures and names of amino acids found in
proteins
The ionization properties of amino acids and peptides
The methods for separation and analysis of proteins

Quiz: 3/23 (Mon)


Chap 2 and 3