Three-Dimensional Structure of

Proteins
Rotation around the -Carbon in a
Polypeptide
A Sterically Nonallowed
Conformation
 The Helix and Pleated Sheet
Conformationally
allowable
structures where
backbone is
optimally H-
bonded (linear H-
bonds).
Pleated Sheet:
Anti-parallel or
Helix (3.613 Helix): parallel
3.6 residues/turn 2.0 residues/turn
Rise = 0.15 nm/ 0.34 nm/residue
residue (anti-parallel) or
13-atom hydrogen- 0.32 nm/residue
bonded loop (parallel)

Linus Pauling and Linus Pauling and

Robert Corey, 1950 Robert Corey, 1951
Antiparallel and Parallel Pleated
Sheets
 Other Secondary Structures

Helix (4.416 Helix):

310 Helix: 4.4 residues/turn
3 residues/turn 0.12 nm/residue
0.20 nm/residue 16-atom hydrogen-
10-atom bonded loop
hydrogen-bonded
loop
Idealized Helices
Hydrogen Bonding Patterns for
Different Helices
Ramachandran Plot

G.N. Ramachandran, 1963

 Fibrous Proteins*

Proteins with an elongated or filamentous form, often

dominated by a single type of secondary structure over a
large distance.

Most fibrous proteins are associated with

connective tissue and help provide
mechanical strength to the tissue.

*
vs. globular proteins
 Structure of Keratin and Keratin-Type
Intermediate Filaments

Keratin is a
principal
component of hair,
horn, nails and
feathers.

Adjacent polypeptide
chains also crosslinked
by disulfide bonds.

Disulfide bond patterns

between are what
determine whether
human hair is straight
or curly.
Coiled-Coil -Helical Dimer of
Keratin

Amphipathic helices:
Residues a, d, a and d
hydrophobic, other
residues hydrophilic.
 Structure of Silk Fibroin

Silk made by
silkworms and
spiders.

Composed of
microcrystalline
array of antiparallel
pleated sheets
where each strand
has alternating Gly
and Ala or Ser
residues.
 Structure of Collagen Fibers
Collagen is the
most abundant
vertebrate protein
and the major
stress-bearing
component of
connective tissue
(bone, teeth,
cartilage, tendon)
and fibrous matrix
of skin and blood
vessels.

3 intertwined left-
handed helices
3.3 residues/turn
Repeating Gly-X-Y
(X often Pro, Y
often Pro or
hydroxyPro)
 The Collagen Triple Helix (Tropocollagen)

Tropocollagen with Gly

Interactions between strands Ala substitution (yellow)
G.N. Ramachandran, 1955
Post-Translational Modifications in
Collagen

Collagen contains unusual, oxidized and

crosslinked lysine residues.

Lysyl oxidase is the enzyme that oxidizes

lysine residues to the aldehyde allysine, which
then forms the crosslinks.

Hydroxyproline is also found in collagen. (Some

lysine residues also hydroxylated.) The enzyme
required for hydroxylation of proline residues is prolyl
hydroxylase, a vitamin C-dependent enzyme.

Scurvy is caused by reduced hydroxyproline in

collagen as a result of vitamin C deficiency.
Biosynthesis and Assembly of Collagen
 Globular Proteins

Proteins with a compact folded structure (with an interior

and exterior), generally containing different types of
secondary structure elements as well as irregular regions.

The vast majority of proteins are globular.

Ribbon Diagram Showing Secondary
Structures in a Globular Protein
 Some Globular Protein Structures

Hemoglobin
(complex of 4
Myoglobin polypeptide
chains or
subunits)

Triose phosphate isomerase (complex of 2 subunits) 20S Proteasome (complex of 28 subunits)

 Additional Elements of Structure:
Turns
R2 often Pro
R3 never Pro

Most common
type of turn

R2 often Pro R3 never Pro

turns turn

trans-Pro (above) or cis-Pro (in Type VI turns) often found in turns.

 Turns with cis-Proline: Type VI turns

Type VIa turn

Type VIb turn

 cis-trans Isomerization of Proline
Residues

Peptidyl-prolyl cis-trans isomerases (rotamases) accelerate the isomerization.

 Additional Elements of Structure:
Loops
Irregularly structured elements
More disordered and flexible than turns
Connects secondary structure elements
Variable in length and shape
Frequently form binding sites and enzyme active sites

The N- and C-terminal arms of proteins are also generally more disordered and irregularly
structured.
 Domain and Motifs in Globular
Proteins: Supersecondary Structure
Some Common Motifs Found in Proteins
hairpin

motif motif

barrels
barrel
Solving 3-D Structure of Proteins

X-ray crystallography
Must crystallize protein.
Structure solved is of protein packed in
crystal and not protein in solution; however,
protein crystals usually have high water
content (and so solution-like in structure).
Almost no limit to size of protein.

Nuclear magnetic resonance

(NMR)
Solution structure.
Can be used to look at dynamics.
Only possible at this time for proteins with
a Mr of < ~50,000.
X-Ray Crystallography Used to Solve First
3-D Protein Structures

Max Perutz with

model of hemoglobin
and John Kendrew
with model of
myoglobin in 1962
 X-Ray Crystallography to Solve
Protein Structures

Protein crystals

3-D
structure
of protein

X-ray
diffraction Electron
pattern density map
Nuclear Magnetic Resonance (NMR)
Comparison of X-Ray and NMR Structures
of Bovine Pancreatic Trypsin Inhibitor

Both X-ray crystallography and

NMR methods usually yield very
similar structures.

Crystallized proteins generally

adopt a conformation very similar
to the averaged NMR solution
structure.
 Molecular Motion in Proteins

Proteins are not static structures but are highly

dynamic:
Breathing - molecular-scale vibrations and
oscillations
Larger-scale conformational changes in both
secondary structural elements and whole domains
 Looking at Protein Dynamics

NMR or lower-resolution methods (e.g., circular

dichroism, absorption spectroscopy, fluorescence
spectroscopy, hydrogen exchange MS) are useful
for looking at dynamics in solution.

X-ray crystallography is not suited for looking at

dynamics, despite being the most powerful
method for determining basic structure. However,
if you can make separate crystals with protein in
different conformations (e.g., protein bound to
ligand or substrate vs. unbound protein), you can
compare structures of both states.
Protein Folding and Stability
Thermodynamics of Folding: Factors
to Consider

Conformational entropy - opposes

protein folding, must be compensated for
by:

The Hydrophobic Effect - favors protein

folding
Internal weak interactions:
Charge-charge interactions (salt bridges)
Hydrogen bonds
van der Waals interactions
 Distribution of Hydrophobic and
Hydrophilic Residues

Cytochrome c

Red: hydrophobic

Green: hydrophilic
 The Hydrophobic Effect in Protein
Folding

Dissolving a hydrophobic group in water solvent forces the water

molecules to form an ordered, low-entropy (clathrate) cage around
the hydrophobic group.

However, entropy of water increases when hydrophobic residues

separate from the water environment and become buried in the
interior of protein.

Since hydrophobic residues separate spontaneously from water and

form proteins interior, location of hydrophobic residues in sequence
will help to determine structure of folded protein.
Hydrophobicity of Different Amino
Acid Sides

Hydropathy scales
Hydropathic Index Plot for Bovine
Chymotrypsinogen
Hydrogen Bonding in a Typical
Protein

Interior of a protein is
mainly a hydrophobic
environment.
Partial or full charges of
polar groups in the interior
are neutralized.
Can involve backbone/
backbone, backbone/side
chain and side chain/side
chain interactions.
Formation of backbone/
backbone hydrogen bonds to
neutralize backbone partial
charges in the interior of a
protein a major factor driving
formation of secondary
structure in the first place.

Lysosyme
 Prediction of Secondary Structure
from Primary Structure (Sequence)
Prediction of Secondary Structure

Adenylate kinase (N-terminal 24 residues)

Bovine pancreatic
trypsin inhibitor
Thermal Denaturation of Ribonuclease
A
 The Protein Folding Problem:
Levinthals Paradox
Ribonuclease A (124 residues) can potentially
form about 1050 conformations. If it tries a
different conformation every 10-13 seconds, it
would take 1050/1013 = 1037 seconds or ~1030
years to try all conformations, yet ribonuclease
A folds in ~1 minute.
There must be pathways of folding with
sequential, dependent steps (intermediates),
instead of a random sampling of all possible
conformations.
Energy Surfaces to Visualize Protein
Conformations
Thermal Denaturation of Bovine
Pancreatic Trypsin Inhibitor
Refolding and Disulfide Bond
Formation
 How to Form the Right Disulfide
Bonds: Protein Disulfide Isomerase
The GroEL-GroES Chaperonin

Other molecular
chaperones:
heat shock
proteins (HSPs)
 The Prion Protein: Protein Folding
and Disease
Prion proteins can exist in
two main conformations,
one of which causes prion-
based diseases such as
Mad Cow Disease.
 Levels of Protein Structure

Primary Structure - amino acid sequence in a

polypeptide
Secondary Structure - local spatial
arrangement of a polypeptides backbone
atoms (without regard to side chain
conformation)
Tertiary Structure - three-dimensional structure
of entire polypeptide
Quaternary Structure - spatial arrangement of
subunits of proteins composed of multiple
polypeptides (protein complexes)
The Four Levels of Protein Structure
 Protein Complexes Terminology

Dimers (2 subunits), trimers (3 subunits),

tetramers (4 subunits), etc.
Polymers - longer, linear or helical multimers

Homotypic: e.g., homodimer (homotypic

dimer) - dimer made up of two identical
polypeptide chains
Heterotypic: e.g., heterodimer (heterotypic
dimer) - dimer made up of two distinct
polypeptide chains
Symmetries of Protein Quaternary
Structures
Prealbumin Dimer

Two-fold cyclic (C2) symmetry;

isologous interactions
Polymeric Helical Proteins
A Dimer without Symmetry

Heterologous asymmetric binding