Chapter 4 The Three-Dimensional Structure of Proteins

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

H for H bonds in protein ≈ H for H bonds with water S > 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 : rotation angle of N-C : rotation angle of C-C

= 180 (or -180)

Ramachandran Plot

Rotation of peptide chain -180 << 180 = 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 vs.

4.2 Protein Secondary Structure

Secondary structure

Local conformation of polypeptide helix, sheet : 60% of the polypeptide chain Random coils and - turn

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

= -45 to -50 = -60

Side chains point outward

Helix

Amino Acid Sequence Affects Helix Stability

Amino acids destabilizing helixElectrostatic repulsion

Glu, Lys, Arg

Bulkiness & shape of adjacent R groups Asn, Ser, Thr, Cys

Restricted rotation Pro

No N-C ()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 Helix Stability

Interaction between amino acid residues at the ends of the helical segment and the electric dipole of helix (+) charged a.a near C-terminus (-) charged a.a near N-terminus

Constraints for the stability of -helix

Intrinsic propensity of a.a to form -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 -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 and 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 structureOverall 3D arrangement of all atoms in a

protein Quaternary structure

Arrangement of protein subunits Classification by higher structure

Fibrous proteinsSingle type of secondary structureProvide support, shape, and external protection

Globular proteinsSeveral types of secondary structureEnzymes and regulatory proteins

Fibrous Proteins

Characteristics of fibrous proteinsStrength and flexibilityWater insoluble

High concentration of hydrophobic amino acids

keratin

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

Coiled coil (left handed twist) of -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 : chain Right-handed supertwist of 3 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, 1950’s)

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; -- loop,

- corner

In general, 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 All / : and segments are interspersed or alternate + : 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 chains and two chains Dimer of 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

“Levinthal’s Paradox”

Hierarchical folding From local folding ( helix, sheets) to entire protein

folding Molten globule state model (hydrophobic

collapse) Initiation of folding by spontaneous collapse by

hydrophobic interactions

Models for Protein Folding

Thermodynamics of Protein Folding

Free-energy funnelUnfolded states

High entropy and high free energy

Folding processDecrease 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

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

Neurodegenerative disorders The amyloid formation is the common phenomenon observed in

various neurodegenerative disorders, including Parkinson’s disease,

Alzheimer’s disease, Huntington’s chorea, Amyotrophic lateral

sclerosis, Prion disease, etc.

Parkinson’s disease

Alzheimer’s disease Prion disease [mad cow disease]

Misfolded protein

Foldedstate

degradation

Accumulation

Amyloid formation

Degenerative disorders

refolding

Amino acids

Amyloid- peptide in Alzheimer’s disease

Creutzfeldt-Jakob disease[Spongiform encephalopathies]