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)

Dihedral angles of : C-N-Ca-C
Dihedral angles of : N-Ca-C-N
 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
 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 Å along the axis, 3.6 amino acids
 y = -45 to -50 f = -60
 Side chains point outward

Linus Pauling Robert Corey

a Helix
 Amino Acid Sequence Affects a Helix
Stability

 Amino acids destabilizing a helix

 Electrostatic repulsion
 Glu, Lys, Arg
 Bulkiness of adjacent R groups
 Asn, Ser, Thr, Cys
 Restricted rotation
 Pro
 No N-Ca 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
 Between n and n+3 (sometimes
n+4) amino acids
 Ion pair
 (+) charged a.a. with (-) charged a.a
 Hydrophobic interaction
 Aromatic amino acids
 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
  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 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
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
 Measured by CD spectra
 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 superhelical twist 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
 Silk Fibroin

 Produced by insects and spiders

  conformation
 Rich in Ala and Gly
 Close packing 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, 1950’s)
 Oxygen carrier in
muscle : containing
heme group
 153 a.a
 Diverse Tertiary Structure of Globular
Proteins
 Small proteins
 Less hydrophobic core (Larger surface area)
 Fewer weak interactions
 Stabilization by covalent bonds (e.g. disulfide bonds)
 Common Structural Patterns

 Motifs, folds, or supersecondary

structures
 Stable arrangements of several
elements of secondary structure
 Domains
 Stable, globular units
 Folding of polypeptide
 Creation of two layers
 Burial of hydrophobic R groups
 -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
 Levels
 Class  fold  evolutionary relationships
 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
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
 Limits to the Size of Proteins

 Genetic coding capacity

 Multimeric proteins
 Generating large structures using small genes
 Viral capsids, cytoskeleton
 Most proteins with Mr >100,000 are multimeric
proteins
 The accuracy of the protein biosynthesis
process
 Error frequency: 1/ 10,000 a.a. addition
 The bigger the protein, the higher the
chance for mistake
4.4 Protein Denaturation and
Folding
 Intrinsically Disordered Proteins

 Contain protein segments that lack definable

structure
 Composed of amino acids whose higher
concentration forces less-defined structure
 Lys, Arg, Glu, and Pro
 Disordered regions can conform to many
different proteins, facilitating interaction
with numerous different partner proteins
Intrinsically Disordered Proteins
P53 can control cell division:
- Unstructured region of C-
terminus interact with four
other proteins.
 Proteostasis
Maintenance of cellular protein activity is accomplished by the
coordination of many different pathways.
 Protein Stability and Folding

• A protein’s function depends on its 3D-structure

• Loss of structural integrity with accompanying loss of
activity is called denaturation
• Proteins can be denatured by:
• heat or cold
• pH extremes
• organic solvents
• chaotropic agents: urea and guanidinium
hydrochloride
 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
 Models for protein folding
 Hierarchical folding
 From local folding (a helix,
 sheets) to entire protein
folding
 Molten globule state model
 Initiation of folding by
spontaneous collapse by
hydrophobic interactions
Proteins folding follow a distinct path
 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
 Protein Folding and Diseases
 Cystic fibrosis
 Misfolding of cystic fibrosis transmembrane conductance
regulator (CFTR)
 Neurodegenerative diseases
 Alzheimer’s, Parkinson’s, Huntinton’s 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
Protein misfolding is the basis of numerous
human diseases
 Molecular Chaperones
 Molecular chaperones
 Proteins facilitating protein folding
 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
Chaperones prevent misfolding

Nucleotide
exchange factor
Chaperonins facilitate folding
GroEL= Hsp60 family
Protein Folding by DnaK and DnaJ
 Chaperonin in Protein Folding

 그림 수정 필요

 Two chambers of the GroEL/Hsp60 alternate in the

binding and facilitated folding of client proteins