Chapter 4

The Three-Dimensional
Structure of Proteins
4.1 Overview of Protein Structure
 Protein Conformation

Conformation
Spatial arrangement of atoms in a protein
Tendency to have the lowest Gibbs free energy
(highest stability)
Noncovalent interactions determining protein
conformation
Maximum hydrogen bonding within the protein
DH for H bonds in protein DH for H bonds with water
DS > 0 by H bonding in protein caused by decrease in
solvation shell of structured water
Hydrophobic interaction
Hydrophobic residues are buried in the protein interior
Ionic interactions (salt bridge)
Disulfide bonds
Native proteins
Proteins in any of their functional, folded
conformation
 The Peptide Bond is Rigid and Planar

Double bond character of peptide bond

Resonance between the carbonyl oxygen and the amide
nitrogen

6 atoms of the peptide group lie in a single plane

No free rotation of peptide C-N bond (trans)
Rotation of peptide chain
f : rotation angle of N-Ca
y : rotation angle of Ca-C

f, y = 180 (or -180)

 Ramachandran Plot

Rotation of peptide chain

-180 < f &y < 180
f, y = 0
Reference point for describing
the angels of rotation
Two peptide bonds are in the
same plane
Restricted by steric overlap
Ramachandran Plot
Plotting of the allowed values
of f vs. y
 4.2 Protein Secondary Structure

Secondary structure
Local conformation of polypeptide
a helix, sheet : 60% of the polypeptide chain
Random coils and - turn
 a Helix
Hydrogen bond between carbonyl O (n) and amid H (n+4)
Right-handed helix
One turn: 5.4 along the axis, 3.6 amino acids
y = -45 to -50 f = -60
Side chains point outward
 Amino Acid Sequence Affects a Helix
Stability
Amino acids destabilizing a helix
Electrostatic repulsion
Glu, Lys, Arg
Bulkiness & shape of adjacent R groups
Asn, Ser, Thr, Cys
Restricted rotation
Pro
No N-Ca (f)rotation kink
No H in N for hydrogen bonding
Flexible rotation
Gly
Tendency to form coil structure different from a helix
 Amino Acid Sequence Affects a Helix
Stability
Interaction between amino acid
residues at the ends of the helical
segment and the electric dipole of a
helix
(+) charged a.a near C-terminus
(-) charged a.a near N-terminus
 Constraints for the stability of a-helix

Intrinsic propensity of a.a to form a-helix

Interactions between side chains
Bulkiness of adjacent side chains
Occurrence of Pro and Gly residues
Interactions between a. a at the ends of the helical
segment and the electric dipole inherent to the a-
helix
 Conformation

stand
Zigzag polypeptide
backbone
sheet, -pleated sheet
Hydrogen bonding between
adjacent strands
Parallel
Antiparallel
Amino acids for specific
sheet structure
Stacking of sheet
-keratins (silk fibroin, spider
web)
Rich in small amino acids:
Gly, Ala
 Turns

Connecting elements
1/3 of amino acids in a globular protein
Turns and loops
turns
Connecting the ends of two adjacent segments of antiparallel
sheet
180o turns involving 4 amino acids and hydrogen bonding
Gly : small and flexible
Pro : cis configuration amenable to a tight turn
 Bond Angles of Amino Acid Content of
Secondary Structure

Relatively restricted range of y and

f depending on the types of
secondary structure
Different distribution of amino acids
in different secondary structures
Circular dichroism
4.3 Protein Tertiary and
Quaternary Structure
 Higher Protein Structure

Tertiary structure
Overall 3D arrangement of all atoms in a
protein
Quaternary structure
Arrangement of protein subunits
Classification by higher structure
Fibrous proteins
Single type of secondary structure
Provide support, shape, and external protection
Globular proteins
Several types of secondary structure
Enzymes and regulatory proteins
 Fibrous Proteins

Characteristics of fibrous proteins

Strength and flexibility
Water insoluble
High concentration of hydrophobic amino acids
 a keratin

Structural protein for hair, wool, feathers, nails, hooves, horns

Providing strength
Coiled coil (left handed twist) of a-helix with hydrophobic amino
acids (A, I, V, M, F)
Forming fibers by hydrophobic interactions
 Disulfide bonds
The more S-S bonds the harder the structure
Permanent wave
Reducing of disulfide bond Generation of new disulfide bond
 Collagen
Providing strength in connective tissue
Tendon, cartilage, organic matrix of bone, cornea
Structure
Left-handed helix with 3 a.a./turn : a chain
Right-handed supertwist of 3 a chains
Amino acid composition
Repeating tripeptide unit, Gly-X, Y
X; Pro, Y; 4-Hyp
35% Gly, 11% Ala, 21% Pro and 4-Hyp
Gly is essential for the structure
Mutation genetic disease
Very low nutritional value
Very close packing
Collagen fibrils
Crosslinking of collagen molecules
by involving lysine, hydroxylysine, histidine
 Silk Fibroin
Produced by insects and spiders
conformation
Rich in Ala and Gly
Close packing of -sheets and interlocking alignment of
R groups
Stabilization by hydrogen bonding and van der Waals
interactions
Flexible

Strand of fibroin emerging

from the spinnerets of a
spider
 Globular Proteins
Globular proteins
Compact
Structural diversity to
carry out diverse
functions

Myoglobin
Structure determined
by x-ray diffraction
studies (John
Kendrew, 1950s)
Oxygen carrier in
muscle : containing
heme group
153 a.a
 Diverse Tertiary Structure of Globular
Proteins
Shared properties w/ myoglobin
Compact folding
Hydrophobic side chains in the interior
Hydrophilic sided chains on the surface
Stabilization by non-covalent interactions
 Common Structural Patterns

Motifs (folds or supersecondary

structures); folding pattern
Stable arrangements of several
elements of secondary structure

Domains
Stable, globular units; distinct functions
 Rules of common protein folding
patterns

Rules of common protein folding

pattern
Hydrophobic interaction
Burial of hydrophobic R groups
Layers of 2nd structures; -a- loop,
a-a corner
In general, a helices and sheets
are in different structural layers
Stacking of the adjacent polypeptide
segments
No crossover connection
conformation is most stable with
slight right-handed twist
Constructing Large Motifs form
Smaller Ones
 Classification of Protein Structures

Structural classification of proteins (SCOP) database

Classification
All a
All
a/ : a and segments are interspersed or alternate
a + : a and regions are segregated
< 1,000 different folds or motifs

Protein family
Proteins with similarities in
Primary sequence
(and/or) Structure
Function
Superfamily
Families with little primary sequence similarity but with
similarities in motifs and function
Tracing structural motifs using protein database
Useful to identify evolutionary relationships
(protein 3rd structure is more conserved than A. a. sequence)
Structural classification from SCOP
database
Structural classification from SCOP
database
Structural classification from SCOP
database
 Quaternary Structure

Hemoglobin
Tetramer : two a chains and two chains
Dimer of a protomer
Symmetric patterns of multimeric proteins with
identical subunits
Rotational symmetry
Cyclic symmetry
Single axis for rotation : Cn , n fold rotation axis
Dihedral symmetry
Intersecting twofold rotational axis and n fold axis at right
angles : Dn, 2n protomers
Icosahedral symmetry
12-cornered polyhedron with 20 equilateral triangular faces
Virus coats and capsids
Helical symmetry
Capsid of tobacco mosaic virus
Actin filaments
Symmetric patterns of multimeric
proteins

Helical symmetry
4.4 Protein Denaturation and
Folding
 Protein Denaturation

Denaturation
A loss of three-dimensional structure
sufficient to cause loss of function
Not necessarily means complete unfolding
or random conformations
Abrupt unfolding over a narrow
temperature range
Cooperative unfolding process
Denaturing agents
Heat
Affect weak interactions (H bonds)
pH
Alternation of the protein net charge
Electrostatic repulsion, disruption of H
bonds
Organic solvents (alcohol, acetone), urea,
guanidine HCl, detergents
Disruption of hydrophobic interactions
 Amino Acid Sequenc Determines
Tertiary Structure

Renaturation
Reversal of denaturation
Amino acid sequence
contains all the information
required to protein folding
First experimental evidence
by Christian Anfinsen
(1950s)
Denaturation of ribonuclease
with urea and reducing agent
Spontaneous refolding to an
active form upon removal of
the denaturing reagents
 Protein Folding

Protein folding in living cells

Not a random, trial-and-error process
E. coli : make 100 a.a. protein in 5 sec
10 possible conformations/ a.a. 10100 conformations
10-13 sec for each conformation 1077 years to test all the
conformations

Levinthals Paradox
 Models for Protein Folding

Hierarchical folding
From local folding (a helix, sheets) to entire protein
folding
Molten globule state model (hydrophobic
collapse)
Initiation of folding by spontaneous collapse by
hydrophobic interactions
 Thermodynamics of Protein Folding

Free-energy funnel
Unfolded states
High entropy and high
free energy
Folding process
Decrease in the number
of conformational
species (entropy) and
free energy
Semistable folding
intermediates
 Molecular Chaperones
Molecular chaperones
Proteins facilitating protein folding by interacting with partially
or improperly folded proteins
Classes of molecular chaperones
Hsp70
Induced in stressed cells (heat shock protein)
Binding to hydrophobic regions of unfolded proteins, preventing
aggregation
Cyclic binding and release of proteins by ATP hydrolysis and
cooperation with co-chaperones (Hsp40 etc.)
E. coli: DnaK (Hsp70), DnaJ (Hsp40)
Chaperonin
Protein complex providing microenvironments for protein folding
E. coli : 10~15% protein require GroES (lid) and GroEL
Isomerases in protein folding
Protein disulfide isomerase (PDI)
Shuffling disulfide bonds
Peptide prolyl cis-trans isomerase (PPI)
Interconversion of the cis and trans isomers of Pro peptide bonds
Protein Folding by DnaK and DnaJ
Chaperonin in Protein Folding
 Protein Folding and Diseases
Cystic fibrosis
Misfolding of cystic fibrosis
-
transmembrane conductance
regulator (CFTR; Cl channel)
Neurodegenerative diseases
Alzheimers, Parkinsons, Huntintons desease, ALS
Prion diseases
Mad cow disease (bovine spongiform encephalopathy, BSE)
Kuru, Creutsfeldt-Jakob disease in human
Scrapie in sheep
Prion : proteinaceous infectious only protein
PrPSc (scrapie) prion form converts PrPC to PrPSc
Neurodegenerative disorders
The amyloid formation is the common phenomenon observed in
various neurodegenerative disorders, including Parkinsons disease,
Alzheimers disease, Huntingtons chorea, Amyotrophic lateral
sclerosis, Prion disease, etc.

Parkinsons disease

Alzheimers disease Prion disease [mad cow disease]

 degradation

Folded Misfolded Amino acids

state protein
refolding

Accumulation

Amyloid formation

Degenerative disorders
Amyloid- peptide in
Alzheimers disease
 Creutzfeldt-Jakob disease
[Spongiform encephalopathies]