Chapter Four

The Three-Dimensional
Structure of Proteins

Paul D. Adams • University of Arkansas

cengage.com/chemistry/campbell
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

Protein Structure
• Many conformations are possible for proteins:
• Due to flexibility of amino acids linked by peptide
bonds

• At least one major conformations has biological

activity, and hence is considered the protein’s native
conformation (folded shape of protein)(spatial
arrangement of atoms that can result from
movement of atoms without breaking a bonds)
 McKee, J. and McKee, T. (2003). Biochemistry: The
Molecular Basis of Life, 3e. McGraw-Hill Companies Inc
4

Proteins
• are polymers composed of sub-units called amino
acids that are linked together by a peptide bond
(amide bond)

• serves as primary structural components of muscles,

connective tissues, hairs, nails, and other parts of a
living organism

• also serves as catalyst in several biochemical

reactions, material transport, and metabolism
regulators in the body, as well as in providing
defenses for the body
 McKee, J. and McKee, T. (2003). Biochemistry: The
Molecular Basis of Life, 3e. McGraw-Hill Companies Inc
5

Functions of Proteins
1. Catalysis
• catalysts hasten chemical reactions without affecting the
nature of the reactants
• enzymes are organic catalysts that are primarily made of
proteins
• examples: hexokinase (glycolysis); nitrogenase (nitrogen
fixation); rubisco or ribulose bisphospate carboxylase
(photosynthesis)

2. Structure
• provide protection and support
• examples: collagen (connective tissues); fibroin (silk
protein); elastin (elastic fibers), found in several tissues in
the body i.e. blood vessels and skin
 McKee, J. and McKee, T. (2003). Biochemistry: The
Molecular Basis of Life, 3e. McGraw-Hill Companies Inc
6

Functions of Proteins
3. Movement
• proteins participate in cell movements
• examples: actin, tubulin, and other composition of the
cytoskeleton
• cytoskeletal proteins are active in cell division,
endocytosis, exocytosis, and amoeboid movement of
WBC

4. Defense
• examples: keratin (skin) aids in protection against mechanical
and chemical injury; fibrinogen and thrombin (bloodclotting)
prevent blood loss when blood vessels are damaged;
immunoglobins (antibodies) are produced by lymphocytes
during invasion of foreign organisms (i.e. bacteria)
 McKee, J. and McKee, T. (2003). Biochemistry: The
Molecular Basis of Life, 3e. McGraw-Hill Companies Inc
7

Functions of Proteins
5. Regulation
• hormone molecules, or a growth factor may bind to cognate
receptors on a target cell may change cellular function
• insulin and glucagon (blood glucose levels)
• growth hormones such as
• somatotrophin is produced in the pituitary gland and it
accelerates transport of amino acids thus promoting production
of proteins
• thyroid stimulating hormones, TSH, are produced in the thydroid
gland and stimulates the production of thyroxine
• thyroxine controls the body’s metabolic rate thus responsible for
the amount of energy used and protein production
• growth factors such as
• bone morphogenic proteins (BMP) – induce formation of bone
and cartilage
• fibroblast growth factor (FGF) – involved in angiogenesis, wound
healing, and embryonic development
 McKee, J. and McKee, T. (2003). Biochemistry: The
Molecular Basis of Life, 3e. McGraw-Hill Companies Inc
8

Functions of Proteins
6. Transport
• many proteins function as carriers of molecules or ions
across membranes and between cells
• hemoglobin carries O2 to the tissues from the lungs
• myoglobin carries O2 to the muscles from the lungs
• lipoproteins: LDL and HDL transport lipids from the liver
and intestines to other organs

7. Storage
• serve as reservoir of essential nutrients
• ovalbumin in bird eggs and casein in mammalian milk are
rich in nitrogen sources during development
• ferritin stores iron
 McKee, J. and McKee, T. (2003). Biochemistry: The
Molecular Basis of Life, 3e. McGraw-Hill Companies Inc
9

Functions of Proteins
8. Stress Response
• proteins aid living organisms in withstanding abiotic
stress for survival
• cytochrome P450 converts toxic substances into less
toxic derivatives
• metallothionein (cysteine rich protein) binds to and
takes away toxic metals
• heat shock proteins (hsp) promote the correct
refolding of damaged proteins at excessively high
temperatures
Levels of Protein Structure
• 1°structure: the sequence of amino acids in a
polypeptide chain, read from the N-terminal end to
the C-terminal end
• 2°structure: the ordered 3-dimensional
arrangements (conformations) in localized regions of
a polypeptide chain; refers only to interactions of the
peptide backbone
• e. g., -helix and -pleated sheet
• 3˚ structure: 3-D arrangement of all atoms
• 4˚ structure: arrangement of monomer subunits with
respect to each other
1˚ Structure
• The 1˚ sequence of proteins determines its 3-D
conformation

• Changes in just one amino acid in sequence can alter

biological function, e.g. hemoglobin associated with
sickle-cell anemia (amino acid =glutamic acid is
substituted by valine in the β chain)
2˚ Structure
• 2˚ of proteins is hydrogen-
bonded arrangement of
backbone of the protein
• Two bonds have free
rotation:
1) Bond between -carbon
and amino nitrogen in
residue
2) Bond between the -
carbon and carboxyl
carbon of residue
• Phi and psi angles of the peptide backbone.
• Definition of the angles that determine the conformation
of a polypeptide chain. The rigid planar peptide groups
(called “playing cards” in the text) are shaded. The angle
of rotation around the Ca–N bond is designated ϕ (phi),
and the angle of rotation around the Ca–C bond is
designated Ψ (psi). These two bonds are the ones around
which there is freedom of rotation.
-Helix
• Coil of the helix is clockwise or right-handed
• There are 3.6 amino acids per turn
(meaning amino acid side chains that are three or four residues apart are bought
together in space and so α-helices are stabilized by hydrogen bond formation
between the carbonyl oxygen of one amino acid, and the amide proton of another
amino acid four residues further along the peptide chain)
• Repeat distance is 5.4Å (Armstrong)
• 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 away
• C=O----H-N hydrogen bonds are parallel to helical
axis
• All R groups point outward from helix
-Helix
(Cont’d)
-Helix (Cont’d)
• Several factors can disrupt an -helix
• proline creates a bend because of (1) the restricted
rotation due to its cyclic structure and (2) its -amino
group has no N-H for hydrogen bonding
• strong electrostatic repulsion caused by the proximity
of several side chains of like charge, e.g., Lys and Arg
or Glu and Asp
• steric crowding caused by the proximity of bulky side
chains, e.g., Val, Ile, Thr
-Pleated Sheet
• Polypeptide chains lie adjacent to one another; may
be parallel or antiparallel
• R groups alternate, first above and then below plane
• Each peptide bond is s-trans and planar
• C=O and N-H groups of each peptide bond are
perpendicular to axis of the sheet
• C=O---H-N hydrogen bonds are between adjacent
sheets and perpendicular to the direction of the sheet
-Pleated Sheet (Cont’d)
-Pleated Sheet (Cont’d)
-Pleated Sheet (Cont’d)
-bulge- a common non-repetitive irregular 2˚ motif in
anti-parallel structure
Structures of Reverse Turns

• Glycine found in reverse turns

• Spatial (steric) reasons
• Polypeptide changes direction
• Proline also encountered in reverse turns. Why?
-Helices and -Sheets
• Supersecondary structures: the combination of -
and -sections, as for example
•  unit: two parallel strands of -sheet connected by
a stretch of -helix
•  unit: two antiparallel -helices
• -meander: an antiparallel sheet formed by a series of
tight reverse turns connecting stretches of a
polypeptide chain
• Greek key: a repetitive supersecondary structure
formed when an antiparallel sheet doubles back on
itself
• -barrel: created when -sheets are extensive enough
to fold back on themselves
Schematic Diagrams of Supersecondary
Structures
Some -Barrel Arrangements
Collagen Triple Helix
• Consists of three polypeptide chains wrapped around
each other in a ropelike twist to form a triple helix
called tropocollagen; MW approx. 300,000
• 30% of amino acids in each chain are Pro and Hyp
(hydroxyproline); hydroxylysine also occurs
• Every third position is Gly and repeating sequences
are X-Pro-Gly and X-Hyp-Gly
• Each polypeptide chain is a helix but not an -helix
• The three strands are held together by hydrogen
bonding involving hydroxyproline and hydroxylysine
• With age, collagen helices become cross linked by
covalent bonds formed between Lys and His residues
The Triple Helix of Collagen

A triple helix. Poly (Gly!Pro!Pro)

is a collagen-like right-handed triple helix
composed
of three left-handed helical chains.
Fibrous Proteins
• Fibrous proteins: contain polypeptide chains
organized approximately parallel along a single axis.
They
• consist of long fibers or large sheets
• tend to be mechanically strong
• are insoluble in water and dilute salt solutions
• play important structural roles in nature
• Examples are
• keratin of hair and wool
• collagen of connective tissue of animals including
cartilage, bones, teeth, skin, and blood vessels
Globular Proteins
• Globular proteins: proteins which are folded to a
more or less spherical shape
• they tend to be soluble in water and salt solutions
• 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
• nearly all have substantial sections of -helix and -
sheet
Comparison of Shapes of Fibrous and
Globular Proteins
3˚ Structure of Proteins

• The 3-dimensional arrangement of atoms in the

molecule.

• In fibrous protein, backbone of protein does not fall

back on itself, it is important aspect of 3˚ not specified
by 2˚ structure.

• In globular protein, more information needed. 3k

structure allows for the determination of the way
helical and pleated-sheet sections fold back on each
other.

• Interactions between side chains also plays a role.

Forces Involved in 3˚ Structure
• Noncovalent interactions, including
• hydrogen bonding between polar side chains, e.g., Ser
and Thr
• hydrophobic interaction between nonpolar side chains,
e.g., Val and Ile
• electrostatic attraction between side chains of opposite
charge, e.g., Lys and Glu
• electrostatic repulsion between side chains of like
charge, e.g., Lys and Arg, Glu and Asp
• Covalent interactions: Disulfide (-S-S-) bonds
between side chains of cysteines
Forces That Stabilize Protein Structure
3°and 4°Structure
• Tertiary (3°) structure: the arrangement in space of
all atoms in a polypeptide chain
• it is not always possible to draw a clear distinction
between 2°and 3°structure

• Quaternary (4°) structure: the association of

polypeptide chains into aggregations
• Proteins are divided into two large classes based on
their three-dimensional structure
• fibrous proteins
• globular proteins
Determination of 3°Structure
• X-ray crystallography
• uses a perfect crystal; that is, one in which all
individual protein molecules have the same 3D
structure and orientation
• exposure to a beam of x-rays gives a series diffraction
patterns
• information on molecular coordinates is extracted by a
mathematical analysis called a Fourier series
• 2-D Nuclear magnetic resonance
• can be done on protein samples in aqueous solution
Large Numbers of Data Points Needed to
Determine 3˚ of a Protein
Large Numbers of Data Points Needed to
Determine 3˚ of a Protein
Large Numbers of Data Points Needed to
Determine 3˚ of a Protein
Myoglobin
• A single polypeptide chain of 153 amino acids
• A single heme group in a hydrophobic pocket
• 8 regions of -helix; no regions of -sheet
• Most polar side chains are on the surface
• Nonpolar side chains are folded to the interior
• Two His side chains are in the interior, involved with
interaction with the heme group
• Fe(II) of heme has 6 coordinates sites; 4 interact with
N atoms of heme, 1 with N of a His side chain, and 1
with either an O2 molecule or an N of the second His
side chain
The Structure of Myoglobin and the Heme
Group
O2 and CO Binding to the Heme group of
Myoglobin
• The oxygen-binding site of
myoglobin. The porphyrin ring
occupies four of the six
coordination sites of the
Fe(II). Histidine F8 (His F8)
occupies the fifth
coordination site of the iron.
Oxygen is bound at the sixth
coordination site of the iron,
and histidine E7 lies close to
the oxygen.
• Oxygen and carbon monoxide binding to the heme
group of myoglobin. The presence of the E7 histidine
forces a 120° angle to the oxygen or CO.
Denaturation and Refolding
• Noncovalent interactions that stabilize proteins are weak
and can be disrupted.
• Denaturation: the loss of 3˚ structural order…”Unfolding”
• Denaturation can be brought about by:
• heat
• large changes in pH, which alter charges on side
chains, e.g., -COO- to -COOH or -NH+ to -NH
• detergents such as sodium dodecyl sulfate (SDS)
which disrupt hydrophobic interactions
• urea or guanidine, which disrupt hydrogen bonding
• mercaptoethanol, which reduces disulfide bonds
Denaturation of a Protein
Denaturation and Refolding in
Ribonuclease

Several ways to denature

proteins
• Heat
• pH
• Detergents
• Urea
• Guanadine hydrochloride
Quaternary Structure of Proteins
• Quaternary (4°) structure: the association of
polypepetide monomers into multisubunit proteins.
Some common multi-subunits include:
• dimers
• trimers
• tetramers

• Noncovalent interactions
• electrostatics, hydrogen bonds, hydrophobic
Oxygen Binding of Hemoglobin (Hb)
• A tetramer of two -chains (141 amino acids each)
and two -chains (153 amino acids each); 22
• Each chain has 1 heme group; hemoglobin can bind
up to 4 molecules of O2
• Binding of O2 exhibited by positive cooperativity;
when one O2 is bound, it becomes easier for the next
O2 to bind
• The function of hemoglobin is to transport oxygen
• The structure of oxygenated Hb is different from that
of unoxygenated Hb
• H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG)
affect the ability of Hb to bind and transport oxygen
Structure of Hemoglobin
Oxygen-Binding to Hb vs. Mb Can Be Shown
Graphically

A comparison of the oxygen

binding
behavior of myoglobin and
hemoglobin. The
oxygen-binding curve of
myoglobin is
hyperbolic, whereas that of
hemoglobin is
sigmoidal. Myoglobin is
50% saturated with
oxygen at 1 torr partial
pressure; hemoglobin
does not reach 50%
saturation until the partial
pressure of oxygen reaches
26 torr.
Conformation Changes That Accompany Hb
Function
• Structural changes occur during binding of small
molecules
• Characteristic of allosteric behavior
• Hb exhibits different 4˚ structure in the bound and
unbound oxygenated forms
• Other ligands are involved in cooperative effect of Hb
can affect protein’s affinity for O2 by altering structure
The Structures of Oxy- and Deoxyhemoglobin

(a) deoxyhemoglobin and (b) oxyhemoglobin

There is much less room at the center of

oxyhemoglobin.
The Effect of H+ on the Binding Affinity of O2
for Hb: The Bohr Effect
• H+ and CO2 bind to Hb and affect the binding affinity of
Hb for O2.

The general features of the Bohr effect. In actively metabolizing

tissue, hemoglobin releases oxygen and binds both CO2 and H+.
In the lungs, hemoglobin releases both CO2 and H+ and binds
oxygen.
Summary of the Bohr Effect
Allosteric Effects on Hb Activity
BPG
(2,3-
bisphosphoglycerate) is
an important
allosteric effector of
hemoglobin.
Protein Folding Dynamics
• Can 3˚ structure of protein be predicted? Yes, within
limitations
• The integration of biochemistry and computing has led
to bioinformatics
• Protein structure prediction is one of the principal
application of bioinformatics
• First step to predict protein structure is to search for
sequence homology
Predicting Protein Structure

• Biochemistry and computing

together gave rise to the field of
bioinformatics

• Predicting protein architecture

is a search of databases of
known structures for sequence
homology

• Homology- Refers to similarity

of two or more sequences
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 inside away from water, and
polar side chains on outside accessible to aqueous
environment
• Hydrophobic interactions are spontaneous
• An introduction to this concept has been described in
the description of the formation of liposomes
Schematic Diagram of a Liposome
Hydrophobic and Hydrophilic Interactions in
Proteins
Unfavorable Entropy Can Occur if Water Introduced to
Hydrophobic Environments: Ordered Arrays of Solvents

Water molecules surround a

hydrophobic molecule and are able to interact with
many more water molecules, reducing entropy
The Importance of Correct Folding
• Proteins that do not fold correctly may interact with
other proteins in an undesired manner and
aggregates may result.
Protein Folding Chaperones
• In the protein-dense environment of a cell, proteins
may begin to fold incorrectly or may associate with
other proteins before folding is completed

• Special proteins called chaperones aid in the correct

and timely folding of many proteins

• hsp70 were the first chaperone proteins discovered

• Chaperones exist in organisms from prokaryotes to

humans
Balancing The Components of Hb