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

 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

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

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

b Turns Connecting elements 1/3 of amino acids in a globular protein Turns and loops b turns Connecting the ends of two adjacent segments of antiparallel b 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

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 b conformation Rich in Ala and Gly Close packing of b-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 Hydrophobic interaction Burial of hydrophobic R groups Layers of 2nd structures; b-a-b loop, a-a corner In general, a helices and b sheets are in different structural layers Stacking of the adjacent polypeptide segments No crossover connection b 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 b a/b : a and b segments are interspersed or alternate a + b : a and b 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 b chains Dimer of ab 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

“Levinthal’s Paradox” 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”

Models for Protein Folding Hierarchical folding From local folding (a helix, b 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 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 Folded state degradation Misfolded protein Folded state Amino acids refolding Accumulation Amyloid formation Degenerative disorders

Amyloid-b peptide in Alzheimer’s disease

Creutzfeldt-Jakob disease [Spongiform encephalopathies]