Chapter 8

Three Dimensional Structures of

Proteins
Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Protein Structure
4 levels of protein structure
Primary structure: aa sequence
Secondary structure: regular chain
organization pattern
Tertiary structure: 3D complex folding
Quarternary structure: association
between polypeptides

Primary Structure
Amino acid sequence determines primary structure
Unique for each protein; innumerable possibilities
Gene sequence determines aa sequence
Each aa is called a residue; numbering (&
synthesis) always from NH2 end toward COOH
end
Amino acids covalently attached to each other by
an amide linkage called as a peptide bond.

Peptide Bond
Peptide bonds are rigid (no free rotation around it) and
planar (2 -C and -O=C-N-H- in one plane)
Partial double bond character due to resonance structures of
peptide bond (bond length is 1.32 Ao instead of 1.49 Ao
(single) or 1.27 Ao (double)
Partial sharing of 2 electron pairs between C and N of
peptide bond
Due to steric hindrance, all peptide bonds in proteins are in
trans configuration
The 2 bonds around the -carbon have freedom of rotation
making proteins flexible to bend and fold

The Peptide Bond is Planar

Partial Double Bond

Trans Configuration

Secondary Structure
Secondary structure is the initial folding pattern
(periodic repeats) of the linear polypeptide.
Secondary structure refers to relative
arrangement of adjacent amino acids
3 main types of secondary structure: -helix, sheet and bend/loop
Secondary structures are stabilized by hydrogen
bonds

The -helix
The -helix is right-handed or clock-wise coil of the
aa chain around a central longitudinal axis. (for Lisoforms left-handed helix is not viable due to steric
hindrance)
The R groups of aa protrude outward from the helix
Each turn has 3.6 aa residues and is 5.4 Ao high
The helix is stabilized by H-bonds between N-H and
C=O groups of every 4th amino acid

-helices can wind around each other to form coiled

coils that are extremely stable and found in fibrous
structural proteins such as keratin, myosin (muscle
fibers) etc

Right-handed helix

Right- and Lefthanded helices

Structure

Stability of -helix
Interactions between aa side chains can influence -helix
stability
Glu-rich sequence destabilizes -helix at pH 7 because of
repulsion between charges
Adjacent lys and / or arg are also unstable
A 3-aa separation between unlike charges is ideal for
ionic interaction and imparts stability
Bulky aa such as asn, cys, ser in close proximity do not
favor -helix (also because of their shape).
Pro and gly do not participate in -helix. Pro has a rigid
N-C bond since N is part of a ring. Poly-gly stretches
are more stable in an alternate coiled structure because of
greater flexibility

Stability of -helix
The peptide bond has a small electric
dipole because of it partial double bond
character
This imparts a partial + charge to the Nterminus of the -helix ; and a negative
charge to the C-terminus
The overall helix dipole is enhanced by
lack of H-bonding at the 4 terminal aa on
each end
Thus, - charged aa at N-end and +
charged aa at C-end have stabilizing
effect

-Pleated Sheet
Extended stretches of 5 or more aa are called -strands
-strands organized next to each other make -sheets
If adjacent strands are oriented in the same direction (N-end
to C-end), it is a parallel -sheet, if adjacent strands run
opposite to each other, it is an antiparallel -sheet. There can
also be mixed -sheets
H-bonding pattern varies depending on type of sheet
R-groups protrude outward from the pleats. Interactions
between R-groups influence stability. Gly and ala common

-sheets are usually twisted rather than flat

Fatty acid binding proteins are made almost entirely of sheets

-Turn / Bend / Loop

Polypeptide chains can fold upon themselves by 180o
forming a bend or a loop.
Usually 4 aa are required to form the turn
H-bond between carbonyl of the 1st aa and aminogroup H of 4th aa
2nd and 3rd aa do not participate in H-bond, but
facilitate the turn Gly and Pro favor b-turns because of
small size and ability to assume cis-configuration
Bends are usually on the surface of globular proteins

-Turn / Bend / Loop

Relative Probability for Amino Acids to occur in

Type of Secondary Structure

Circular dichroism spectra to assess Secondary Structures

Motifs
Also called supersecondary structure
Motifs are very stable folding patterns of
secondary structure.
They are combinations of helices, sheets,
bends etc.
such motifs are seen repeatedly in many
different proteins.
Many kinds of motifs are possible and are
given unique names such as -barrel, zinc
fingers, leucine zippers etc.
Multiple motifs make up protein domains

Some Motifs

Motifs

-sheets
arranged as a
barrel

Common in fatty
acid binding
proteins

Small motifs are assembled into

larger motifs

Tertiary Structure
3-D arrangement of amino acids
Polypeptides undergo folding or bundling up.
Includes interactions between amino acids that are
far apart in primary structure
The folding creates pockets and sites for binding
substrates, ligands and cofactors
Folding is such that non-polar residues are buried
inside, polar residues are exposed outwards to
aqueous environment.

Tertiary Structure
5 kinds of bonds stabilize tertiary structure: Hbonds, van der waals interactions, hydrophobic
interactions, ionic interactions and disulphide
linkages
In disulphide linkages, the SH groups of two
neighboring cysteines form a S-S- bond called as a
disulphide linkage. It is a covalent bond, but readily
cleaved by reducing agents that supply the protons to
form the SH groups again
Reducing agents include -mercaptoethanol and DTT

Reduction of Disulfide Linkages

Protein Denaturation
The information for every proteins 3-D folded
structure is inherent in its sequence
The naturally occuring pattern of protein folding is
called its native conformation
The unfolding or loss of protein native conformation
is called protein denaturation
Changes in temperature, pH, salt concentration and
presence of organic solvents or urea and detergents
cause protein denaturation
Denaturation is reversible; removal of disrupting
agents cause renaturation. Most proteins renature
spontaneously, others require assistance.

Co-operativity in Protein Denaturation

Molecular Chaperones
Sometimes protein folding is facilitated by other proteins
called molecular chaperones.
Chaperones often associate with a polypeptide chain as it is
being synthesized and guide it to fold in the right
functionally active conformation.
This usually needs some input of energy in the form of
ATP.
Two classes of chaperones: Heat-shock proteins, Hsp70,
are found in cells stressed by heat. They bind to denatured
proteins and prevent incorrect folding or aggregation
Chaparonins: multi-polypeptide complexes that facilitate
folding by an elaborate mechanism

Errors in Protein Folding

Various genetic disorders may be the result of
incorrect protein folding
The most common cause of cystic fibrosis is due to
misfolding of the CFTR protein resulting from a
single amino acid deletion in the primary structure.
Mad cow disease and other forms of spongiform
encephalopathies, (also called as Prion diseases:
proteinaceous infection only) result from misfolding
of the Prion Protein (PrP) in the brain. Normally
folded PrP is converted to misfolded PrP by
interaction with the latter.

Globular Proteins
Polypeptides chains are folded into a
spherical or globular shape
Consist of several regions of different
secondary structures
Most enzymes and regulatory proteins are
globular
Examples: myoglobin, albumin, globulin,
lysozyme, ribonuclease

Structure of Myoglobin

Structural Domains
Many proteins are organized into multiple domains
Domains are compact globular units that are connected
by a flexible segment of the polypeptide lacking
secondary/tertiary structure
Domains usually have biological activity. Each
domain contributes a specific function to the overall
protein
Different proteins may share similar domain
structures, eg: kinase-, cysteine-rich-, globin-domains

Multi-Domain
Structure of
Ca-ATPase

Domains of c-Src protein

SH3 domain = ~ 60 AA
SH2 domain = ~100AA
SH3 domain= ~300 AA kinase domain
(phosphotransferase)

Fibrous Proteins
Polypeptides chains are arranged in long sheets
or strands
Consist of one predominant secondary structure
Fibrous proteins are structural proteins: their
functions include providing shape and
mechanical strength
Examples: collagen, -keratin, silk fibrion

-Keratin

Collagen

Silk Fibroin

Quaternary Structure
Association of more than one polypeptides
Each unit of this protein is called as a subunit and
the protein is an oligomeric protein
Subunits (monomers) can be identical or different
The protein is homopolymeric or
heteropolymeric
Disulfide bonds usually stabilize the oligomer.
Electrostatic / hydrophobic interactions may also
contribute to quaternary structure stability.

Quaternary Structure of Hemoglobin

Methods for the 3-D Structure of a Protein

NMR spectroscopy and X-ray

crystallography are the two most
commonly used methods to
determine the 3-D structures of
macromolecules.

Principles of NMR
Nuclei act as small bar
magnets and possess a net
magnetic moment

Bo

Precession at
the Larmor
frequency

Application of
external Bo

=
Precession around
external Bo
No Magnetic Field
Isotropic conditions

Very homogeneous
Magnetic field

Principles of NMR
One-dimensional NMR
HSQC

chemical shift

1H,15N

15N

Two-dimensional NMR

Three-dimensional NMR

1H

chemical shift

NMR Spectroscopy

What is crystallography ?
The light microscope uses
visible light to view the objects
as long as not generally
smaller than its wavelength
(400 -700 nm)
Similarly, crystallography can
be used to view the objects
as small as 0.1 nm using the
monochromatic X-rays with
the similar wavelength range.

Why are crystals used?

1. X-ray scattering from a single molecule would

be incredibly weak and impossible to detect.
2. A crystal arranges a huge number of molecules
in the same orientation, so that scattered waves
can add up in phase and raise the signal to a
measurable level.

Principles of X-ray crystallography

X-ray Diffraction

1.9

Fourier theory
1. The diffraction pattern is related to the
object diffracting the waves through a
mathematical operation called the
Fourier transform.
2. So the real image (electron electron
density) can be obtained by by taking
the Fourier transform as long as both
the amplitude and the phase
information is known

What is electron density?

A map of the distribution of electrons in
the molecule, i.e. an electron density map.
However, since the electrons are mostly
tightly localized around the nuclei, the
electron density map is a pretty good
picture of the molecule.

Protein X-Ray Crystallography

Crystallization

X-ray Data

Structure Determination Refinement and Analysis

Crystallization Process
1. For obtaining crystals, the molecules must
assemble into a periodic lattice.
2. Start with a solution of the protein with a fairly high
concentration (2-50 mg/ml) and add reagents that
reduce the solubility close to spontaneous
precipitation (called superstaturated state).
3. With slowing further concentration, and under
conditions suitable for the formation of a few
nucleation sites, crystals may start to grow.
4. Many conditions are generally tested usually by
initial screening, then followed by a systematic
optimization of conditions.

Phase Diagram for Crystallization Experiment

State A: Protein stays undersaturated and no crystals grow

State B: Protein crystallizes and the concentration of protein
in solution drops to saturation
State C: Protein precipitates, but crystals may still grow

Crystallization Methods
1. Batch Method
2. Vapour Diffusion Method

i) Hanging Drop
ii) Sitting Drop
iii) Sandwich Drop
3. Dialysis Method

Batch Method
The precipitant and protein are mixed directly
This can be done in a glass vial or under oil

Vapour Diffusion:Hanging Drop Method

Vapour Diffusion: Sitting Drop Method

Dialysis Method
Protein is equilibrated against a larger volume of
precipitant through a dialysis membrane.

X-ray crystallography requires a single

crystal as the sample

But all you get is precipitate. Need to try more conditions.

X-ray crystallography requires a single

crystal as the sample

A good quality crystal is grown for data collection!!!

Protein Structure determination

HIS-374

Fe
HIS-313

ASP-315
Water
TRP-389

TYR-310

MET-299
TYR-303

Final Model of HIF-PH2 based on X-ray Crystallography

Ribbon Diagram of Crystal Structure of HIF-PH2

Ordered
amino acids
188 to 403
represented as
a ribbon
diagram
Mixed /
structure with
strand core
folded into a
jellyroll motif

Chapter 8

Protein Function: Hemoglobin in

Microcosm
Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Protein Function

Reversible binding of ligands is essential

Specificity of ligands and binding sites

Ligand binding is often coupled to conformational changes, sometimes

quite dramatic (Induced Fit)
In multisubunit proteins, conformational changes in one subunit can affect
the others (Cooperativity)
Interactions can be regulated

Illustrated by:
Structures of myoglobin and hemoglobin
Oxygen-binding curves
Regulation of O2 binding

Functions of Proteins
Storage of ions and molecules
myoglobin, ferritin
Transport of ions and molecules
hemoglobin, serotonin transporter
Defense against pathogens
antibodies, cytokines
Muscle contraction
actin, myosin
Biological catalysis
chymotrypsin, lysozyme

Prosthetic Groups
Some proteins have non-peptide components as
part of the functional unit. These non-peptide
components are called as prosthetic groups.
Prosthetic groups are permanently associated with
the protein and are required for protein function
Proteins with prosthetic groups are called
complex or conjugated proteins. otherwise they
are simple proteins.

Ligands
A molecule that binds reversibly to a protein is called
its ligand. Ligands may be small peptides or nonpeptide molecules. Physiological ligands are usually
endocrine, metabolic or environmental factors.
Ligand binding is a trigger for the activation of protein
function.
Ligand binding usually results in a conformational
change in the proteins 3-D structure.
The ligand binding site on the protein is
complementary to the ligand in size, shape and charge.
Ligand binding is specific.

Binding: Quantitative Description

Consider a process in which a ligand (L)

binds reversibly to a site in the protein (P)

ka
P

kd

PL

The kinetics of such a process is described by:

the association rate constant ka
the dissociation rate constant kd

After some time, the process will reach the equilibrium

where the association and dissociation rates are equal
The equilibrium composition is characterized by the the
equilibrium constant Ka

k a [P] [L] = k d [PL]

ka
[PL]
Ka =
=
[ P ] [ L] k d

How does Le Chatliers

Principle apply?

ka
[PL]
Ka =
=
[ P ] [ L] k d

[L]n
% ligand binding sites occupied = = -------------------[L]n + Kd
100%

50%

Lots of ligands, equilibrium

pushed very far to the right

Few ligands,
Equilibrium not pushed
very far to right

Think of this as % of ligand binding sites occupied.

Hyperbolic Curve for Ligand Binding

Oxygen-Binding Proteins
Myoglobin, Hemoglobin, Cytochromes bind O2.
Oxygen is transported from lungs to various tissues
via blood in association with hemoglobin
In muscle, hemoglobin gives up O2 to myoglobin
which has a higher affinity for O2 than hemoglobin.
Cytochromes participate is redox reactions.
The [O2] is dependent on its partial pressure pO2.
For O2 binding, = pO2/(pO2 + P50), where P50 is
the pO2 at half saturation binding.

Oxygen-binding curve of Myoglobin

The Prosthetic Heme Group

The heme group is responsible for the O2-binding capacity of
hemoglobin and myoglobin.
The heme group consists of the planar aromatic protoporphyrin
made up of four pyrrole rings linked by methane bridges.
A Fe atom in its ferrous state (Fe+2) is at the center of
protoporphyrin.
Fe+2 has 6 coordination bonds, four bonded to the 4 pyrrole N
atoms. The nucleophilic N prevent oxidation of Fe+2.
The two additional binding sites are one on either side of the
heme plane.
One of these is occupied by the imidazole group of His.
The second site can be reversibly occupied by O2, which is
hydrogen-bonded to another His.

Structure of Heme

Interactions
of aa with
heme in
myoglobin

Structure of Myoglobin
The primary structure is its sequence. This consists of 153
aa with Mr = 16,700
Secondary structure: 8 alpha-helical regions designated as
A to H starting at the NH2 terminus. No -sheets.
The tertiary structure: folding of the 8 helices into a single
domain called the globin fold.
Several bends and loops formed in the process; they are
labeled reflecting the helical segments they connect.
Folding results in the formation of a pocket or box where
the heme group fits in. Hydrophobic aa are buried in the
interior, lining the heme pocket.
The tertiary structure is stabilized mostly by hydrophobic
interactions. There are no disulphide bonds.
Myoglobin is monomeric, there is no quarternary structure.

Structure of Myoglobin

Hemoglobin subunits are structurally

Similar to Myoglobin

Sequence Similarity between

Hemoglobin and Myoglobin

Hemoglobin Structure
Hemoglobin is an oligomeric protein made up of 2
dimers, a total of 4 polypeptide chains: 1122.
The (141 aa) and (146 aa) subunits are highly
homologous. Total Mr of hemoglobin is 64,500
Each subunit consists of 7 () or 8 () alpha helices and
several bends and loops folded into a single globin
domain.
Each chain has one side where nonpolar groups are
exposed rather than polar groups. These regions interact
such that the 4 chains are held together by hydrophobic
interactions. H-bonds and ionic interactions also
contribute to quarternary structure.
Each subunit has a heme-binding pocket.

Different forms of Hemoglobin

When hemoglobin is bound to O2, it is called
oxyhemoglobin. This is the relaxed (R ) state.
The form with a vacant O2 binding site is called
deoxy-hemoglobin and corresponds to the tense (T)
state.
If iron is in the oxidized state as Fe+3, it is unable to
bind O2 and this form is called as methemoglobin
CO and NO have higher affinity for heme Fe+2 than
O2 and can displace O2 from Hb, accounting for their
toxicity.

Changes Induced by O2 Binding

O2 binding rearranges electrons within Fe making it more
compact so that it fits snugly within the plane of porphyrin.
Since Fe is bound to histidine of the globin domain, when Fe
moves, the entire subunit undergoes a conformational
change.
This causes hemoglobin to transition from the tense (T) state
to the relaxed (R) state.
Inter-subunit interactions influence O2 binding to all 4
subunits resulting in cooperativity

O2-binding triggers conformational change

Inter-subunit interactions
Two alpha and two beta subunits (1122)
Strong interactions between unlike subunits
About 30 aa involved in inter-subunit interactions;
dimer remains intact after disruption with urea
In deoxyhemoglobin, the C-terminal His and Asp of
-subunits interact with lys of -subunit. Also arg
and asp on C-terminus of one -subunit interact with
those of 2nd -subunit.
In oxyhemoglobin, the histidines have shifted to the
interior and can no longer form ion pairs

T and R states of Hemoglobin

Hemoglobin exists in two major conformational
states: Relaxed (R ) and Tense (T)
R state has a higher affinity for O2.
In the absence of O2, T state is more stable; when O2
binds, R state is more stable, so hemoglobin
undergoes a conformational change to the R state.
The structural change involves readjustment of
interactions between subunits.
The 11 and 22 dimers rearrange and rotate
approximately 15 degrees with respect to each other

T-state to R-state Transition

O2-binding kinetics
4 subunits, so 4 O2-binding sites
O2 binding is cooperative meaning that each subsequent O2
binds with a higher affinity than the previous one
Similarly, when one O2 is dissociated, the other three will
dissociate at a sequentially faster rate.
Due to positive cooperativity, a single molecule is very rarely
partially oxygenated.
There is always a combination of oxygenated and
deoxygenated hemoglobin molecules. The percentage of
hemoglobin molecules that remain oxygenated is represented
by its oxygen saturation
O2-binding curves show hemoglobin saturation as a function
of the partial pressure for O2.

Sigmoidal
Curve for
O2 binding

Oxygen Saturation Curve

Saturation is maximum at very high O2 pressure in the
lungs (pO2 = ~ 100 torr).
As hemoglobin moves to peripheral organs and the O2
pressure drops (pO2 = ~20 torr), saturation also drops
allowing O2 to be supplied to the tissues.
Due to co-operative binding of O2 to hemoglobin, its
oxygen saturation curve is sigmoid.
Such a curve ensures that at lower pO2, small
differences in O2 pressure result in big changes in O2
saturation of hemoglobin. This facilitates
dissociation of O2 in peripheral tissues.

O2-transfer from Hemoglobin to Myoglobin

The myoglobin curve shows us that only at extremely
low O2 pressure, there is no binding of O2 to
myoglobin.
At relatively low pO2, the saturation shoots up and
there is almost complete saturation, meaning that all
molecules are associated with O2.
At the pO2 that myoglobin is fully saturated,
hemoglobin is less than half saturated. This facilitates
the transfer of O2 from hemoglobin to myoglobin in
the muscle.

Effectors of O2 binding
Small molecules that influence the O2-binding capacity
of hemoglobin are called as effectors (allosteric
regulation)
Positive or negative effectors; homotropic or heterotropic
effectors
Oxygen is a homotropic positive effector
Positive effectors shift the O2-binding curve to the left,
negative effectors shift the curve to the right
From a physiological view, negative effectors are
beneficial since they increase the supply of oxygen to the
tissues.

The Bohr Effect

The regulation of O2-binding to hemoglobin by H+ and CO2
is called the Bohr effect
Both H+ and CO2 are negative effectors of O2-binding.
Addition of a proton to His imidazole group at C-terminus of
-subunit facilitates formation of salt bridge between His and
Asp and stabilization of the T quaternary structure of
deoxyhemoglobin.
CO2 reduces O2 affinity by reacting with terminal NH2 to
form negatively charged carbamate groups that form salt
bridges to stabilize deoxyhemoglobin.
Metabolically active tissues need more O2; they generate
more CO2 and H+ which causes hemoglobin to release its O2.

Effect of pH on Oxygen -Binding

2,3-Bisphosphoglycerate
2,3-Bisphosphoglycerate is a negative effector.
A single 2,3-BPG binds to a central pocket of
deoxyhemoglobin and stabilizes it by interacting
with three positively charged aa of each -chain.
2,3-BPG is normally present in RBCs and shifts the
O2-saturation curve to the right
Thus, 2,3-BPG favors oxygen dissociation and
therefore its supply to tissues
In the event of hypoxia, the body adapts by
increasing the concentration of 2,3-BPG in the RBC

Effect of 2,3-Bis-Phosphoglycerate

Fetal Hemoglobin
Fetal hemoglobin has 2 and 2 chains
The chain is 72% identical to the chain.
A His involved in binding to 2,3-BPG is replaced with
Ser. Thus, fetal Hb has two less + charge than adult Hb.
The binding affinity of fetal hemoglobin for 2,3-BPG is
significantly lower than that of adult hemoglobin
Thus, the O2 saturation capacity of fetal hemoglobin is
greater than that of adult hemoglobin
This allows for the transfer of maternal O2 to the
developing fetus.

Sickle-cell anemia is due to

a mutation in hemoglobin
Glu6 Val in the chain of Hb
The new Valine side chain can bind to a
different Hb molecule to form a strand
This sickles the red blood cells
Untreated homozygous individuals
generally die in childhood
Heterozygous individuals exhibit a
resistance to malaria

Formation of Hb Strands
in Sickle-Cell Anemia