Chapter 4 The Three-Dimensional Structure of Proteins.
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Transcript of 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]