Chapter 4

The Three-Dimensional
Structure of Proteins

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Chapter Outline
(4-1) Protein structure and function
(4-2) Primary structure of proteins
(4-3) Secondary structure of proteins
(4-4) Tertiary structure of proteins
(4-5) Quaternary structure of proteins
(4-6) Protein-folding dynamics

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Protein Structure
• Biologically active proteins are polymers that consist
of amino acids linked by covalent peptide bonds
• Many conformations are possible for proteins due to
flexibility of amino acids
• Native conformations: 3-D shapes of proteins with
biological activity
• Many proteins have large segments of random
structure as they have no obvious regular repeating
structure

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Levels of Protein Structure
• Primary structure (1°): Order in which amino acids
are covalently linked together
• Read from the N-terminal end to the C-terminal end
• Secondary structure (2°): Ordered 3-D arrangement
in space of the backbone atoms in a polypeptide
chain
• Tertiary structure (3°): 3-D arrangement of all atoms
in a protein, including those in side chains and in
prosthetic groups
• Quaternary structure (4°): Arrangement of subunits
with respect to one another
• Subunits: Individual parts of a large molecule
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Primary Structure
• Amino acid sequence helps determine the 3-D
conformation of a protein, which determines its
properties
• Determination of this sequence is a routine operation
in classical biochemistry
• Changes in one amino acid residue in a sequence
can alter biological functions
• Example - Hemoglobin associated with sickle-cell
anemia

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Secondary Structure
• Hydrogen-bonded arrangement of the polypeptide
chain
• Peptide chains are linked at opposite corners by
swivels
• Each amino acid residue has two bonds with free
rotation that are designated by the Ramachandran
angles, phi (Φ) and psi (Ψ)
• Bond between the α-carbon and amino nitrogen of that
residue
• Bond between the α-carbon and carboxyl carbon of
that residue
• Types - α-helix and β-pleated sheet arrangements
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Figure 4.1 - Peptide Conformation

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α-Helix
• Stabilized by hydrogen bonds parallel to the helix
axis within the backbone of a single polypeptide
chain
• Helical conformation allows for a linear arrangement
• Provides maximum bond strength and makes the
conformation stable
• Features
• Coil of the helix is clockwise or right-handed
• There are 3.6 residues for each turn of the helix
• Linear distance between corresponding points on
successive turns (pitch) is 5.4 Å

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α-Helix (continued 1)
• Each peptide bond is s-trans and planar
• C═O of each peptide bond is hydrogen bonded to the
N—H of the fourth amino acid residue
• C═O≡H—N hydrogen bonds are parallel to the
helical axis
• All R groups point outward from the helix

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Figure 4.2 - Four Different Graphic Representations
of the α-Helix

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Factors That Disrupt the α-Helix
• Bending of the polypeptide backbone because of
proline
• Restricts rotation due to its cyclic structure
• Results in absence of the N—H for hydrogen bonding
in the α-amino group
• Strong electrostatic repulsion caused by the proximity
of several side chains of like charges
• Examples - Lys and Arg or Glu and Asp
• Steric repulsion, or crowding, caused by the proximity
of bulky side chains
• Example - Val, Ile, and Thr

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β-Pleated Sheet
• Hydrogen bonds between peptide chains can be
interchain or intrachain
• Give rise to a zigzag structure and are perpendicular to
the direction of the protein chain
• Polypeptide chains lie adjacent to one another
• If chains run in the same direction, the sheet will be
parallel
• If chains run in the opposite direction, the sheet will be
antiparallel
• R groups alternate above and below the plane
• Each peptide bond is s-trans and planar

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Figure 4.4 - Hydrogen Bonding in β-Pleated
Sheets

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Figure 4.5 - Three-Dimensional Form of the Antiparallel
β-Pleated Sheet Arrangement

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Irregularities in Regular β-Pleated Structures
• Found in shorter stretches than with the α-helix
• Sometimes break up the regular nature of the α-helix
• β-bulge: Common nonrepetitive irregular secondary
motif in antiparallel β-sheets
• Occurs between two normal β-structure hydrogen
bonds and involves two residues on one strand and
one on the other

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Figure 4.6 - β-Bulges

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Reverse Turns
• Parts of proteins where the polypeptide chain folds
back on itself
• Contain glycine
• Formed because of spatial (steric) reasons
• Result
• Polypeptide chain changes direction
• Proline is also encountered in reverse turns because
of its cyclic structure

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Figure 4.7 - Structures of Reverse Turns

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Supersecondary Structures and Domains
• Supersecondary structures - Result from the
combination of α- and β-strands
• βαβ unit - Two parallel strands of β-sheet are
connected by a stretch of α-helix
• αα unit - Contains two antiparallel α-helices
• Also called helix-turn-helix
• β-meander - Antiparallel sheet is formed by a series of
tight reverse turns connecting stretches of the
polypeptide chain
• Greek key - Formed when a polypeptide chain doubles
back on itself

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Figure 4.8 - Schematic Diagrams of
Supersecondary Structures

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Supersecondary Structures and Domains
(continued)

• Motifs: Repetitive supersecondary structures

• β-barrel - Created when β-sheets are extensive
enough to fold back on themselves
• Provide information about the folding of proteins
• Do not help predict the biological function of the
protein
• Proteins that have the same type of function have
similar protein sequences
• Domains with similar conformations are associated
with a particular function

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Figure 4.9 - Some β-Barrel Arrangements

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Collagen Triple Helix
• Organized in water-insoluble fibers of great strength
• Fibers consist of three polypeptide chains wrapped
around each other in a ropelike twist, or triple helix
• Called tropocollagen (MW = 300,000)
• Each chain has a repeating sequence of three amino
acid residues, X—Pro—Gly or X—Hyp—Gly
• Hyp - Hydroxyproline
• Formed from proline by a specific hydroxylating enzyme
after the amino acids are linked together
• First position (X) can be occupied by any amino acid
• Every third position must be occupied by glycine

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Collagen Triple Helix (continued)
• All three individual collagen
chains are helices that differ
from the α-helix
• All three strands are held
together by hydrogen bonds
involving hydroxyproline and
hydroxylysine
• With age, collagen helices
become cross-linked by
covalent bonds formed by
reactions of Lys and His
residues

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Fibrous Proteins
• Contain polypeptide chains organized approximately
parallel along a single axis
• Consist of long fibers or large sheets
• Tend to be mechanically strong
• Insoluble in water and have the ability to dilute salt
solutions
• Play important structural roles in nature
• Examples
• Keratin in hair and wool
• Collagen in connective tissue, including cartilage,
bones, teeth, skin, and blood vessels

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Globular Proteins
• Proteins in which the backbone folds on itself to
produce a more or less spherical shape
• Most of their polar side chains are on the outside and
interact with the aqueous environment by hydrogen
bonding and ion–dipole interactions
• Most of their nonpolar side chains are buried inside
• Have substantial sections of α-helix and β-sheet
• Tend to be soluble in water and salt solutions
• Have compact structures

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Figure 4.12 - Comparison of the Shapes of
Fibrous and Globular Proteins

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Tertiary Structure of Proteins
• 3-D arrangement of all atoms in a molecule
• Includes the conformations of side chains and the
positons of any prosthetic groups
• In fibrous proteins:
• Backbone of the protein does not fold back on itself
• Arrangement of the atoms of the side chains is not
specified by the secondary structure
• In globular proteins:
• Tertiary structure helps determine the way helical and
pleated-sheet sections fold back on each other
• Interactions between side chains play an essential role
in folding
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Forces Involved in Tertiary Structures
• Tertiary structures depend on noncovalent
interactions
• Backbone hydrogen bonding between polar side
chains of amino acids
• Hydrophobic interaction between nonpolar side chains
• Electrostatic attraction between side chains of opposite
charge
• Complexing several side chains to a single metal ion
• Disulfide bonds between side chains of cysteines
• Restrict folding patterns available to polypeptide chains
• Absent in myoglobin and hemoglobin but present in
chymotrypsin and trypsin

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Figure 4.13 - Forces That Stabilize the Tertiary
Structure of Proteins

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Determination of Tertiary Structure
• X-ray crystallography: Uses a perfect crystal in
which all individual protein molecules have the same
3-D structure and orientation
• When a pure crystal is exposed to a beam of X rays, a
diffraction pattern is produced
• Pattern results when electrons in each atom scatter the
X rays
• Scattered X rays can reinforce or cancel each other
giving a characteristic pattern for each molecule
• Information is extracted by a mathematical analysis
called a Fourier series

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Figure 4.14 (A) - X-Ray Diffraction Photograph of
Glutathione Synthetase

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Determination of Tertiary Structure (continued)
• Nuclear magnetic resonance (NMR) spectroscopy
• In 2-D NMR, large collection of data points are
subjected to a computer analysis
• Uses a Fourier series to analyze results
• Requires considerable amount of computing power
and milligram quantities of protein
• Uses protein samples in aqueous solution

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Figure 4.14 (B) - NMR Data for α-Lactalbumin

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Figure 4.14 (C) - Tertiary Structure of α-
Lactalbumin

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Myoglobin
• Consists of a single polypeptide chain of 153 amino
acid residues and a prosthetic group, heme, in a
hydrophobic pocket
• Heme: Iron-containing cyclic compound
• Has a compact structure
• Carries eight α-helical regions, which are stabilized
by hydrogen bonding in the polypeptide backbone
• β-pleated sheet regions are absent
• Exterior contains most of the polar side chains
• Interior contains nonpolar side chains and two
histidine residues that are involved in interactions
with the heme group and bound O2
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Figure 4.15 - Structure of Myoglobin

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Heme Group
• Presence affects conformation of the polypeptide
• Consists of:
• A metal ion, Fe(II)
• Has six coordination sites and forms six metal–ion
complexation bonds
• Four sites are occupied by N atoms of the four pyrrole-type
rings of the porphyrin
• Fifth coordination site is occupied by one of the N atoms of the
imidazole side chain of histidine residue F8
• O2 is bound at the sixth coordination site
• An organic part, protoporphyrin IX
• Consists of four five-membered rings based on the
pyrrole structure that are linked by methine (—CH═)
groups
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Figure 4.16 - Structure of the Heme Group

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Oxygen: Imperfect Binding to the Heme Group
• More than one molecule
can bind to heme
• Affinity of free heme for
carbon monoxide (CO)
is 25,000 times greater
than its affinity for O2

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Figure 4.18 - Oxygen and Carbon Monoxide Binding to
the Heme Group of Myoglobin

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Denaturation and Refolding
• Denaturation: Unraveling of the 3-D structure of a
macromolecule caused by the breakdown of
noncovalent interactions that can result from:
• Heat
• Large changes in pH, which alter charges on side
chains
• Detergents, such as sodium dodecyl sulfate (SDS),
which disrupt hydrophobic interactions
• Urea and guanidine hydrochloride, which disrupt
hydrogen bonding
• β-mercaptoethanol

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Figure 4.19 - Denaturation of a Protein

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Denaturation and Refolding in Ribonuclease
• β-mercaptoethanol is used to
reduce disulfide bridges to
two sulfhydryl groups
• Urea is added to the reaction
mixture to:
• Facilitate unfolding
• Increase accessibility of the
disulfides to the reducing
agent
• Native conformation can be
recovered by removing
mercaptoethanol and urea
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Quaternary Structure of Proteins
• Pertains to proteins that consist of more than one
polypeptide chain
• Each chain is called a subunit
• Oligomers: Molecules that are made up of a number
of smaller subunits
• Include dimers, trimers, and tetramers
• Chains interact with one another noncovalently via
electrostatic attractions, hydrogen bonds, and
hydrophobic interactions
• Allosteric: Property of multisubunit proteins such that a
conformational change in one subunit induces a drastic
change in another subunit

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Hemoglobin
• Tetramer with:
• Two α-chains
• 141 residues long
• Two β-chains
• 146 residues long
• Overall structure - α2β2
• Most amino acids of the
α-chain, β-chain, and
myoglobin are
homologous

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Oxygen Binding of Hemoglobin (Hb)
• One molecule of
myoglobin binds one O2
• Hemoglobin can bind up
to four molecules of O2
• Binding of O2 to
hemoglobin exhibits
positive cooperativity
• When one O2 is bound, it
becomes easier for the
next O2 to bind

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Myoglobin versus Hemoglobin
• Myoglobin
• Function - Oxygen storage
• Requirement - Bind strongly to O2 at very low
pressures
• Saturation - 50% at 1 torr partial pressure of O2
• Hemoglobin
• Function - Oxygen transport
• Requirement - Bind strongly to O2 and release O2
easily
• Saturation - 100% when O2 pressure is 100 torr

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Structures of Hemoglobin
• Structure of oxygenated hemoglobin is different from
that of deoxygenated hemoglobin

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Conformational Changes That Accompany
Hemoglobin Function
• Ligands involved in cooperative effects when O2
binds to Hb can affect protein affinity for O2 by
altering structure
• H+ and CO2
• Effect of H+ is called the Bohr effect

• Increase in [H+] reduces O2 affinity of Hb and causes

protonation of key amino acids
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Conformational Changes That Accompany
Hemoglobin Function (continued 1)
• Acid–base properties of Hb
• Oxygenated form of Hb is a stronger acid since it has a
lower pKa than its deoxygenated form
• Deoxygenated hemoglobin has higher affinity for H+
than the oxygenated form

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Figure 4.25 - Oxygen Saturation Curves for Myoglobin
and for Hemoglobin at Five Different pH Values

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Conformational Changes That Accompany
Hemoglobin Function (continued 2)
• Hb is also bound to 2,3-
bisphosphoglycerate
(BPG)
• Binding is electrostatic
• In the absence of BPG,
oxygen-binding capacity
of Hb would be higher
• BPG plays a role in the
supply of O2 to the
growing fetus

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Figure 4.27 - Binding of BPG to
Deoxyhemoglobin

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Predicting Protein Structure
• One of the principal
applications of bioinformatics
• Involves searching the
databases of known
structures for sequence
homology
• Similarity of monomer
sequences in polymers
• Methods
• Fold recognition
• De novo prediction
• Modeling algorithms

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Figure 4.33 - Predicted versus Actual Protein
Structures

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Hydrophobic Interactions: A Case Study in
Thermodynamics
• Hydrophobic interactions are major factors in protein
folding
• Folding occurs so that:
• Nonpolar hydrophobic side chains tend to be on the
inside and away from water
• Polar side chains are on the outside and have access
to the aqueous environment
• Example
• Liposomes: Spherical aggregates of lipids arranged
so that the polar head groups are in contact with water
and the nonpolar tails are sequestered from water

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Figure 4.34 - Schematic Diagram of a Liposome

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Figure 4.35 - Three-Dimensional Structure of the
Protein Cytochrome C

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Favorable versus Unfavorable Hydrophobic
Interactions
• Hydrophobic interactions are spontaneous processes
• Result in increase in the entropy of the Universe
ΔS univ > 0

• Example
• Mixing liquid hydrocarbon hexane (C6H14) with water
yields one layer of hexane and one layer of water
• Formation of the mixed solution is nonspontaneous
• Formation of the two layers is spontaneous
• Unfavorable entropy terms arise if solution formation
requires the creation of ordered arrays of solvent

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Favorable versus Unfavorable Hydrophobic
Interactions (continued)
• Results in high degree of order
• Prevents the dispersion of energy, which lowers the
entropy
• Nonpolar substances associate with one another by
hydrophobic interactions and are excluded from water

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The Importance of Correct Folding
• In the protein-dense environment of the cell, proteins
may begin to:
• Fold incorrectly as they are produced
• Associate with other proteins before completing their
folding process

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Protein-Folding Chaperones
• Aid in the correct and timely folding of many proteins
• hsp70 - First chaperone protein to be discovered
• Exist in organisms from prokaryotes to humans
• Example
• α-hemoglobin stabilizing protein (AHSP) maintains a
proper stoichiometry between α-chains and β-chains in
globin
• Prevents α-chains from causing damage to blood cells
• Delivers α-chains to the β-chains

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Figure 4.38 - Balancing the Components of
Hemoglobin

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