4 | The Three-

Dimensional Structure
of Proteins
© 2017 W. H. Freeman and Company
 CHAPTER 4
The Three-Dimensional Structure
of Proteins

Learning goals:
• Structure and properties of the peptide bond
• Structure and function of fibrous proteins
• Structure analysis of globular proteins
• Protein folding and denaturation
 Structure of Proteins

• 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 an entropy cost to folding the protein into
one specific native fold.
 Favorable Interactions in Proteins
• Hydrophobic effect
– The release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy.
• Hydrogen bonds
– Interaction of N−H and C=O of the peptide bond leads to local regular
structures such as  helices and  sheets.
• London dispersion
– Medium-range weak attraction between all atoms contributes
significantly to the stability in the interior of the protein.
• Electrostatic interactions
– long-range strong interactions between permanently charged groups
– Salt bridges, especially those buried in the hydrophobic environment,
strongly stabilize the protein.
Four Levels of Protein Structure
Primary Structure: The Peptide Bond
• The structure of the protein is partially dictated by
the properties of the peptide bond.
• The peptide bond is a resonance hybrid of two
structures.
• The resonance causes the peptide bonds:
– to be less reactive compared with esters, for
example
– to be quite rigid and nearly planar
– to exhibit a large dipole moment in the favored
trans configuration
Resonance in the Peptide Bond
 The Rigid Peptide Plane and
the Partially Free Rotations
• Rotation around the peptide bond is not permitted due
to resonance structure.
• Rotation around bonds connected to the  carbon is
permitted.
The Polypeptide Is Made Up of a Series
of Planes Linked at α Carbons
 Secondary Structures
• Secondary structure refers to a local spatial
arrangement of the polypeptide backbone.
• Two regular arrangements are common:
• the  helix
– stabilized by hydrogen bonds between nearby residues
• the  sheet
– stabilized by hydrogen bonds between adjacent
segments that may not be nearby
• Irregular arrangement of the polypeptide chain is
called the random coil.
 The  Helix
• Helical backbone is held together
by hydrogen bonds between the
backbone amides of an n and
n + 4 amino acids.
• It is a right-handed helix with 3.6
residues (5.4 Å) per turn. n+4
• Peptide bonds are aligned
roughly parallel with the helical
n
axis.
• Side chains point out and are
roughly perpendicular with the
helical axis.
What Is a Right-Handed Helix?
 The  Helix: Top View
• The inner diameter of the helix (no side chains) is
about 4–5 Å.
• too small for anything to fit “inside”
• The outer diameter of the helix (with side chains) is
10–12 Å.
• happens to fit well into the major groove of dsDNA
• Amino acids #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 bond is impossible.
• Gly acts as a helix breaker because the tiny R group
supports other conformations.
• Attractive or repulsive interactions between side
chains 3 to 4 amino acids apart will affect formation.
 Propensity of Amino Acid Residues to Take Up an α-Helical
TABLE 4-2
Conformation
Amino acid ΔΔG° (kJ/mol)a Amino acid ΔΔG° (kJ/mol)a

Ala 0 Leu 0.79

Arg 0.3 Lys 0.63

Asn 3 Met 0.88

Cys 3 Pro >4

Gln 1.3 Ser 2.2

Glu 1.4 Thr 2.4

Gly 4.6 Tyr 2.0

His 2.6 Trp 2.0

Ile 1.4 Val 2.1

Sources: Data (except proline) from J. W. Bryson et al., Science 270:935, 1995. Proline data from J. K.
Myers et al., Biochemistry 36:10,923, 1997.
a
DDG° is the difference in free-energy change, relative to that for alanine, required for the amino acid
residue to take up the α-helical conformation. Larger numbers reflect greater difficulty taking up the α-
helical structure. Data are a composite derived from multiple experiments and experimental systems.
 The Helix Dipole
• Recall that the peptide bond has a strong dipole
moment.
– C−O (carbonyl) negative
– N−H (amide) positive
• All peptide bonds in the  helix have a similar
orientation.
• The  helix has a large macroscopic dipole moment
that is enhanced by unpaired amides and carbonyls
near the ends of the helix.
• Negatively charged residues often occur near the
positive end of the helix dipole.
  Sheets
• The planarity of the peptide bond and tetrahedral
geometry of the  carbon create a pleated sheet-like
structure.
• Sheet-like arrangement of the backbone is held together
by hydrogen bonds between the backbone amides in
different strands.
• Side chains protrude from the sheet, alternating in an
up-and-down direction.
 Parallel and Antiparallel b Sheets
• Multi -strand interactions are called sheets.
• Sheets are held together by the hydrogen bonding
of amide and carbonyl groups of the peptide bond
from opposite strands.
• Two major orientations of  sheets are determined
by the directionality of the strands within:
– Parallel sheets have strands that are oriented in the same
direction.
– Antiparallel sheets have strands that are oriented in
opposite directions.
In parallel  sheets, the H-bonded strands run in the
same direction.
• Hydrogen bonds between strands are bent (weaker).
In antiparallel  sheets, the H-bonded strands run in
opposite directions.
• Hydrogen bonds between strands are linear (stronger).
 b Turns
•  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.
 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.
 Protein Tertiary Structure
• Tertiary structure refers to the overall spatial
arrangement of atoms in a protein.
• Stabilized by numerous weak interactions between
amino acid side chains
BUT largely hydrophobic effect and hydrophobic interaction

• Interacting amino acids are not necessarily next to

each other in the primary sequence.
Forces That Stabilize the Tertiary Structure of
Proteins
 Favorable Interactions in Proteins
• Hydrophobic effect
- All amino acids that contain the non-polar side chains will aggregate
inside of the protein
• London dispersion
– Medium-range weak attraction between all atoms contributes
significantly to the stability in the interior of the protein.
• Hydrogen bonds
– Interaction of N−H and C=O of the peptide bond leads to local regular
structures such as  helices and  sheets.

• Electrostatic interactions
– long-range strong interactions between permanently charged groups
– Salt bridges, especially those buried in the hydrophobic environment,
strongly stabilize the protein.
 Protein Tertiary Structure
• Two major classes:
– fibrous and globular proteins
Fibrous Proteins
 Protein Tertiary Structure
• Tertiary structure refers to the overall spatial
arrangement of atoms in a protein.
• Stabilized by numerous weak interactions between
amino acid side chains
 largely hydrophobic and polar interactions
 can be stabilized by disulfide bonds
• Interacting amino acids are not necessarily next to
each other in the primary sequence.
• Two major classes:
– fibrous and globular (water or lipid soluble)
 Fibrous Proteins:
From Structure to Function

TABLE 4-3 Secondary Structures and Properties of Some Fibrous Proteins

Structure Characteristics Examples of occurrence

α Helix, cross-linked by Tough, insoluble protective α-Keratin of hair, feathers, nails

disulfide bonds structures of varying hardness and
flexibility
β Conformation Soft, flexible filaments Silk fibroin
Collagen triple helix High tensile strength, without stretch Collagen of tendons, bone matrix
Structure of -Keratin in Hair
Chemistry of Permanent Waving
 Structure of Collagen
• Collagen is an important constituent of connective
tissue: tendons, cartilage, bones, cornea of the eye.
• Each collagen chain is a long Gly- and Pro-rich left-
handed helix.
• Three collagen chains intertwine into a right-handed
superhelical triple helix.
• The triple helix has higher tensile strength than a
steel wire of equal cross section.
• Many triple-helices assemble into a collagen fibril.
 Silk Fibroin
• Fibroin is the main protein in silk from moths and
spiders.
• Antiparallel  sheet structure
• Small side chains (Ala and Gly) allow the close
packing of sheets.
• Structure is stabilized by:
– hydrogen bonding within sheets
– London dispersion interactions between sheets
Water-Soluble Globular Proteins
 Motifs (folds)

• Specific arrangement of several secondary

structure elements
– all  helix
– all  sheet
– both
• Motifs can be found as recurring structures in
numerous proteins (different function, similar
folding)
• Globular proteins are composed of different motifs
folded together.
Motifs (folds)
Repeated Motifs Contribute to Final Fold
 Quaternary Structure
A quaternary structure is formed by the assembly of
individual polypeptides into a larger functional cluster.
 Protein Stability and Folding
• A protein’s function depends on its 3D structure.
• Loss of structural integrity with accompanying loss
of activity is called denaturation.
• Proteins can be denatured by:
• heat or cold
• pH extremes
• organic solvents
• chaotropic agents: urea and guanidinium
hydrochloride
 Ribonuclease Refolding Experiment
• Ribonuclease is a small protein that contains eight cysteines
linked via four disulfide bonds.
• Urea in the presence of 2-mercaptoethanol fully denatures
ribonuclease.
• When urea and 2-mercaptoethanol are removed, the protein
spontaneously refolds, and the correct disulfide bonds are
reformed.
• The sequence alone determines the native conformation.
• The experiment is quite “simple” but so important it earned
Chris Anfinsen the 1972 Chemistry Nobel Prize.
 How Can Proteins Fold So Fast?
• Proteins fold to the lowest-energy fold in the
microsecond to second time scales. How can they
find the right fold so fast?
• It is mathematically impossible for protein folding to
occur by randomly trying every conformation until
the lowest-energy one is found (Levinthal’s paradox).
• There must be a shortcuts
• Folding pathway of a large polypeptides is
complicated, however rapid progress has been made
in this field (predict the structure of small
polypeptides
Proteins Folding Follow a Distinct Path
Chaperones Prevent Misfolding and
Aggregation of Unfolded Peptides
Protein Misfolding Is the Basis of
Numerous Human Diseases
• Native (correctly folded) 
amyloid is a soluble globular
protein,
• Misfolded  amyloid promotes
aggregation at newly exposed
protein-protein interface.
• Correctly folded helices are lost
and peptides form  strands, 
helices, and  sheets.
 Chapter 4: Summary

• the two most important secondary structures

–  helices
–  sheets
• how properties and function of fibrous proteins are
related
• how to determine three-dimensional structures of
proteins
• one of the largest unsolved puzzles in modern
biochemistry: how proteins fold