Chapter 4: The Three Dimensional Structure of Proteins

4.1 Overview of Protein Structure

Unlike most organic polymers, protein molecules adopt a specific
three-dimensional conformation.
This structure is able to fulfill a specific biological function
This structure is called the native fold
The native fold has a large number of favorable interactions
within the protein
There is a cost in conformational entropy of folding the protein
into one specific native fold
Favorable Interactions in Proteins
o Hydrophobic effect
Release of water molecules from the structured
solvation layer around the molecule as protein folds
increases the net entropy
Leu, Ile, Val, Phe, Trp
o Hydrogen bonds
Interaction of N-H and C=O of the peptide bond leads
to local regular structures such as -helices and sheets
o London dispersion
Medium-range weak attraction between all atoms
contributes significantly to the stability in the interior
of the protein
o Electrostatic interactions
Long-range strong interactions between permanently
charged groups
Salt-bridges, esp. buried in the hydrophobic
environment strongly stabilize the protein
4 Levels of Protein Structure
o The primary structure consists of a sequence of amino
acids linked together by peptide bonds and includes any
disulfide bonds.
o The resulting polypeptide can be arranged into units of
secondary structure, such as an helix.
o The helix is a part of the tertiary structure of the folded
polypeptide, which is itself one of the subunits that make
up the quaternary structure of the multisubunit protein,
in this case hemoglobin.
Peptide Bond is Rigid and Planar
o Structure of the protein is partially dictated by the
properties of the peptide bond

o The peptide bond is a resonance hybrid of two canonical

structures
o The resonance causes the peptide bonds
to be less reactive compared to esters, for example
to be quite rigid and nearly planar
to exhibit a large dipole moment in the favored trans
configuration
Each peptide bond has some double-bond character
due to resonance and cannot rotate.

The Rigid Peptide Plane and the Partially Free Rotations

o Rotation around the peptide bond is not permitted
o Rotation around bonds connected to the alpha carbon is
permitted
o (phi): angle around the -carbonamide nitrogen bond
o (psi): angle around the -carboncarbonyl carbon bond
o In a fully extended polypeptide, both phi and psi are 180
The polypeptide is made up of a series of planes linked at the carbon
o Three bonds separate sequential carbons in a
polypeptide chain. The NC and CC bonds can rotate,
described by dihedral angles designated and ,
respectively. The peptide CN bond is not free to rotate.
Other single bonds in the backbone may also be
rotationally hindered, depending on the size and charge of
the R groups.
o Phi and Psi angle can be between -180 to +180, but many
values are prohibited by steric interference
o Allowed values for phi and psi angles become evident
when the psi is plotted versus psi in the Ramachandran
plot

Distribution of Phi and Psi dihedral angles

o Some phi and psi combinations are very unfavorable
because of steric crowding of backbone atoms with other
atoms in the backbone or side chains
o Some phi and psi combinations are more favorable
because of chance to form favorable H-bonding
interactions along the backbone
o A Ramachandran plot shows the distribution of phi
and psi dihedral angles that are found in a protein
shows the common secondary structure elements
reveals regions with unusual backbone structure
4.2 Protein Secondary Structure
Secondary Structures
o Secondary structure refers to a local spatial arrangement
of the polypeptide backbone
o Two regular arrangements are common:
o The helix
stabilized by hydrogen bonds between nearby
residues
o The sheet
stabilized by hydrogen bonds between adjacent
segments that may not be nearby
Another is turn
o Irregular arrangement of the polypeptide chain is called the
random coil.
Does not properly describe the structure of
segments.
o The helix
Basics
Helical backbone is held together by hydrogen
bonds between the backbone amides of an n
and n+4 amino acids
Right-handed helix with 3.6 residues (5.4 )
per turn

Peptide bonds are aligned roughly parallel with

the helical axis
Side chains point out and are roughly
perpendicular with the helical axis
Can occur in left handed or right handed form
Internal hydrogen bonding occurs by hydrogen
bond between the hydrogen atom attached to
the electronegative carbonyl oxygen atom of
the fourth amino acid on the amino-terminal
side of that peptide bond.
Dimensions
The inner diameter of the helix (no side chains)
is about 45
o Too small for anything to fit inside
The outer diameter of the helix (with side
chains) is 1012
o Happens to fit well into the major groove
of dsDNA
Residues 1 and 8 align nicely on top of each
other
Sequence affects helix stability
Not all polypeptide sequences adopt -helical
structures
Small hydrophobic residues such as Ala and
Leu are strong helix formers
Pro acts as a helix breaker because the
rotation around the N-Ca bond is impossible,
because it forms a rigid ring, destabilizing kink,
Pro also has no substituent hydrogen to
participate in H-bond
Gly acts as a helix breaker because the tiny
R-group supports other conformations
Attractive or repulsive interactions between
side chains 34 amino acids apart will affect
formation
o Glu residues block because the
negatively charged carboxyl groups of
adjacent Glu residenes repel each other,
same for Lys/Arg rseisdues with positively
charged R groups.
Identity of amino acids near ends
o Negatively charged found near amino
terminus
If positive = destabalizing

o Postively charged found near carboxy

terminus
If negative = destabalziing
The Helix Dipole
Recall that the peptide bond has a strong
dipole moment
Carbonyl O negative
Amide H positive
All peptide bonds in the helix have a similar
orientation
The helix has a large macroscopic dipole
moment
Negatively charged residues often occur near
the positive end of the helix dipole
o Sheets
Basics
The planarity of the peptide bond and
tetrahedral geometry of the -carbon create a
pleated sheet-like structure
Sheet-like arrangement of backbone is held
together by hydrogen bonds between the
backbone amides in different strands
Side chains protrude from the sheet alternating
in up and down direction
Parallel and Antiparallel B sheets
Parallel or antiparallel orientation of two chains
within a sheet are possible
In parallel b sheets the H-bonded strands run in
the same direction
o Resulting in bent H-bonds (weaker)
In antiparallel b sheets the H-bonded strands
run in opposite directions
o Resulting in linear H-bonds (stronger)
o Turns
Basics
turns occur frequently whenever strands in
sheets change the direction
The 180 turn is accomplished over four amino
acids
The turn is stabilized by a hydrogen bond from
a carbonyl oxygen to amide proton three
residues down the sequence

Proline in position 2 or glycine in position 3 are

common in turns
Found near the surface of the protein
Proline Isomers
Most peptide bonds not involving proline are in
the trans configuration (>99.95%)
For peptide bonds involving proline, about 6%
are in the cis configuration. Most of this 6%
involve -turns
Proline isomerization is catalyzed by proline
isomerases
o Trans Cis form
Circular Dichroism (CD) Analysis
Any form of structural asymmetry in a
molecule gives rise to differences in a
absorption of left-handed versus right handed
circularly polarized light.
CD measures the molar absorption difference
of left- and right-circularly polarized light:
= L R
Chromophores in the chiral environment
produce characteristic signals
CD signals from peptide bonds depend on the
chain conformation
Method for assessing common secondary
structure and monitoring folding
4.3 Protein Tertiary and Quaternary Structures
Protein Tertiary Sturcture
o Tertiary structure refers to the overall spatial arrangement
of atoms in a protein
o Stabilized by numerous weak interactions between amino
acid side chains.
Largely hydrophobic and polar interactions
Can be stabilized by disulfide bonds
o Interacting amino acids are not necessarily next to each
other in the primary sequence.
o Two major classes
Fibrous and globular (water or lipid soluble)
Fibrous polypeptide chains arranged in long
strands or sheets (single secondary
structures), insoluble in water

Globular polypeptide chains folded into a

spherical or globular shape (multiple types of
secondary structures)

Collagen
o Collagen is an important constituent of connective tissue:
tendons, cartilage, bones, cornea of the eye
o Each collagen chain is a long Gly- and Pro-rich left-handed
helix
o Three collagen chains intertwine into a right-handed
superhelical triple helix
o The triple helix has higher tensile strength than a steel wire
of equal cross section
o Many triple-helices assemble into a collagen fibril
o The amino acid sequence in collagen is repeating
tripeptide unit of Gly-X-Y where X is Pro and Y is 4-Hyp
o 4-Hydroxyproline in Collagen
Forces the proline ring into a favorable pucker
Offer more hydrogen bonds between the three
strands of collagen
The post-translational processing is catalyzed by
prolyl hydroxylase and requires -ketoglutarate,
molecular oxygen, and ascorbate (vitamin C)
In absence of Vit C cells cannot hydroxylate the Pro
at the Y positions, this leads to collagen instability,
requires enzyme prolyl 4-hydroxylase this enzyme
restores F2+ state
Alpha-Keratin
o Proteins constitute almost entire dry weight of hair, wool,
nails, claws
o Arranged in coiled coil fashion, supertwisting amplifies the
strength of the overall structure
o Rich in hydrophobic residues
o Hair -keratin is an elongated helix. Pairs of these helices
are interwound in a left-handed sense to form two-chain
coiled coils. These then combine in higher-order structures
called protofilaments and protofibrils. About four
protofibrils combine to form an intermediate filament.
o Permanent Waxing
The hair to be waved is first bent around a form of
apprpripate shape.
A solution of reducing agent usually thio containing is
used and heat is applied.
The reducing agent cleaves disulfide bond to form
two cys residues.

Reducing agent is removed and oxidizing agent is

added to establish new disulfide bonds

Silk Fibroin
o Fibroin is the main protein in silk from moths and spiders
o Antiparallel sheet structure
o Small side chains (Ala and Gly) allow the close packing of
sheets
o Structure is stabilized by
hydrogen bonding within sheets
London dispersion interactions between sheets
o Spider Silk
Used for webs, egg sacks, and wrapping the prey
Extremely strong material
stronger than steel
can stretch a lot before breaking
A composite material
crystalline parts (fibroin-rich)
rubber-like stretchy parts
Motifs
o Specific arrangement of several secondary structure
elements
All alpha-helix
All beta-sheet
Both
o Motifs can be found as reoccurring structures in numerous
proteins
o Proteins are made of different motifs folded together
o Example is B-a-B loop or B-barrel
Domain
o Is a part of the polypeptide chain that is independently
stable or could undergo movement as a single entity with
respect to the entire protein
Proteins with significant similarities in primary structure and/or
with similar terityary structure are in the same protein family,
two or more families with little similarities in amino acid
sequence but use the same structural motif are superfamilies.
Quaternary Structure
o Quaternary structure is formed by the assembly of
individual polypeptides into a larger functional cluster
Protein Structure Methods: X-Ray Crystallography
o Steps needed
Purify the protein
Crystallize the protein

 Collect diffraction data

Calculate electron density
Fit residues into density
o Pros
No size limits
Well-established
o Cons
Difficult for membrane proteins
Cannot see hydrogens
Structure Methods: Biomolecular NMR
o Steps needed
Purify the protein
Dissolve the protein
Collect NMR data
Assign NMR signals
Calculate the structure
o Pros
No need to crystallize the protein
Can see many hydrogens
o Cons
Difficult for insoluble proteins
Works best with small proteins
Intrinsically Disordered Proteins
o Contain protein segments that lack definable structure
o Lack a hydrophobic core
o Composed of charged amino acids whose higher
concentration forces less-defined structure
Lys, Arg, Glu, and Pro
o Disordered regions can conform to many different proteins,
facilitating interaction with numerous different partner
proteins
o Protein p27 in cell division, protein p53 in control of cell
division.
4.4 Protein Denaturation and Folding
Proteostasis
o Maintenance of cellular protein activity is accomplished by
the coordination of many different pathways.
o Three kinds of processes contribute to proteostasis, in
some cases with multiple contributing pathways.
First, proteins are synthesized on a ribosome.
Second, multiple pathways contribute to protein
folding, many of which involve the activity of
complexes called chaperones. Chaperones
(including chaperonins) also contribute to the

refolding of proteins that are partially and transiently

unfolded.
Third, finally, proteins that are irreversibly unfolded
are subject to sequestration and degradation by
several additional pathways. Partially unfolded
proteins and protein-folding intermediates that
escape the quality-control activities of the
chaperones and degradative pathways may
aggregate, forming both disordered aggregates and
ordered amyloidlike aggregates that contribute to
disease and aging processes.
Protein Stability and Folding
o A proteins function depends on its 3D-structure
o Loss of structural integrity with accompanying loss of
activity is called denaturation
o Proteins can be denatured by:
heat or cold
pH extremes
organic solvents: alcohol or acetone
chaotropic agents: urea and guanidinium
hydrochloride
o Organic solvents, urea, detergents act by distrupting the
hydrophobic interactions that make up the core of globular
proteins, extremes of pH alter the net charge on the
protein causing electrostatic repulsion and the disturption
of h-bonds.
Ribonuclease Refolding Experiment
o Ribonuclease is a small protein that contains 8 cysteines
linked via four disulfide bonds
o Urea in the presence of 2-mercaptoethanol fully denatures
ribonuclease
o When urea and 2-mercaptoethanol are removed, the
protein spontaneously refolds, and the correct disulfide
bonds are reformed
o The primary amino acid sequence alone determines the
native conformation
o Tm is the midpoint of the temperature range over which
denaturation occurs (melting temepature)
How can proteins fold so fast?
o Proteins fold to the lowest-energy fold in the microsecond
to second time scales. How can they find the right fold so
fast?
o It is mathematically impossible for protein folding to occur
by randomly trying every conformation until the lowestenergy one is found (Levinthals paradox)

o Search for the minimum is not random because the

direction toward the native structure is thermodynamically
most favorable
Protein folding follow a distinct path
o Small regions of secondary structure are assembled first
and then gradually incorporated into larger structures.
Chaperones prevent misfolding
o The chaperones do not actively promote the folding of the
substrate protein, but instead prevent aggregation of
unfolded peptides.
o Hsp70 (heat shock protein) are chaperones
Protect both prteins subject to denaturation by heat
and new peptide molecules being synthesized
Use ATP hydrolysis
o Chaperonins are another type of chaperones
GroEL/GroES
Protein misfolding is the basis of numerous human diseases
o Protein molecules whose normal structure includes regions
of sheet undergo partial folding. In a small number of the
molecules, before folding is complete, the -sheet regions
of one polypeptide associate with the same region in
another polypeptide, forming the nucleus of an amyloid.
Additional protein molecules slowly associate with the
amyloid and extend it to form a fibril.
o This characteristic plaques on the exterior of nervous
tissue in people with Alzheimer disease. The aromatic side
chains shown here play a significant role in stabilizing the
amyloid structure. Amyloid is rich in sheet, with the
strands arranged perpendicular to the axis of the amyloid
fibril. Amyloid- peptide takes the form of two layers of
extended parallel sheet. Some amyloid-forming peptides
may fold to form left-handed -helices.