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CHAPTER 4 Proteins: Structure, Function, Folding Key topics: – Structure and properties of the peptide bond – Structural hierarchy in proteins – Structure and function of fibrous proteins – Protein folding and denaturation – Structure analysis of globular proteins Structure of Proteins • Unlike most organic polymers, protein molecules adopt a specific 3-dimensional conformation in the aqueous solution. • This structure is able to fulfill a specific biological function • This structure is called the native fold • The native fold has a large number of favorable interactions within the protein • There is a cost in conformational entropy of folding the protein into one specific native fold Protein Structures are compact Protein Sci. 2006 August; 15(8): 1829–1834. doi: 10.1110/ps.062305106. Favorable Interactions in Proteins • Hydrophobic effect – Release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy • Hydrogen bonds – Interaction of N-H and C=O of the peptide bond leads to local regular structures such as -helixes and -sheets • London dispersion – Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein • Electrostatic interactions – Long-range strong interactions between permanently charged groups – Salt-bridges, esp. buried in the hydrophobic environment strongly stabilize the protein Structure of the Peptide Bond • Structure of the protein is partially dictated by the properties of the peptide bond • The peptide bond is a resonance hybrid of two canonical structures • The resonance causes the peptide bonds – be less reactive compared to e.g. esters – be quite rigid and nearly planar – exhibit large dipole moment in the favored trans configuration The Rigid Peptide Plane and the Partially Free Rotations • Rotation around the peptide bond is not permitted • Rotation around bonds connected to the alpha carbon is permitted • f (phi): angle around the -carbon—amide nitrogen bond • y (psi): angle around the -carbon—carbonyl carbon bond • In a fully extended polypeptide, both y and f are 180° Distribution of f and y Dihedral Angles • Some f and y combinations are very unfavorable because of steric crowding of backbone atoms with other atoms in the backbone or side-chains • Some f and y combinations are more favorable because of chance to form favorable H-bonding interactions along the backbone 3 • Ramachandran plot shows the distribution of f and y dihedral angles that are found in a protein • shows the common secondary structure elements • reveals regions with unusual backbone structure Ramachandran Plot Secondary Structures • Secondary structure refers to a local spatial arrangement of the polypeptide chain • Two regular arrangements are common: • The helix – stabilized by hydrogen bonds between nearby residues • The sheet – stabilized by hydrogen bonds between adjacent segments that may not be nearby • Irregular arrangement of the polypeptide chain is called the random coil The helix • The backbone is more compact with the y dihedral (N–C—C–N) in the range ( 0 < y < -70) • Helical backbone is held together by hydrogen bonds between the nearby backbone amides • Right-handed helix with 3.6 residues (5.4 Å) per turn • Peptide bonds are aligned roughly parallel with the helical axis • Side chains point out and are roughly perpendicular with the helical axis The helix: Top View • The inner diameter of the helix (no side-chains) is about 4 – 5 Å • Too small for anything to fit “inside” • The outer diameter of the helix (with side chains) is 10 – 12 Å • Happens to fit well into the major groove of dsDNA • Residues 1 and 8 align nicely on top of each other • What kind of sequence gives an helix with one hydrophobic face? Sequence Affects Helix Stability • Not all polypeptide sequences adopt -helical structures • Small hydrophobic residues such as Ala and Leu are strong helix formers • Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible • Gly acts as a helix breaker because the tiny R-group supports other conformations The Helix Macro-Dipole • Peptide bond has a strong dipole moment – Carbonyl O negative – Amide H positive • All peptide bonds in the helix have a similar orientation • The helix has a large macroscopic dipole moment • Negatively charged residues often occur near the positive end of the helix dipole Sheets • The backbone is more extended with the y dihedral (N–C—C–N) in the range ( 90 < y < 180) • The planarity of the peptide bond and tetrahedral geometry of the -carbon create a pleated sheet-like structure • Sheet-like arrangement of backbone is held together by hydrogen bonds between the more distal backbone amides • Side chains protrude from the sheet alternating in up and down direction Parallel and Antiparallel Sheets • Parallel or antiparallel orientation of two chains within a sheet are possible • In parallel sheets the H-bonded strands run in the same direction • In antiparallel sheets the H-bonded strands run in opposite directions Circular Dichroism (CD) Analysis • CD measures the molar absorption difference of left- and right- circularly polarized light: = L – R • Chromophores in the chiral environment produce characteristic signals • CD signals from peptide bonds depend on the chain conformation Turns (Hairpins) -turns occur frequently whenever strands in sheets change the direction • The 180° turn is accomplished over four amino acids • The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence • Proline in position 2 or glycine in position 3 are common in -turns • Proline Isomers • Most peptide bonds not involving proline are in the trans configuration (>99.95%) • For peptide bonds involving proline, about 6-20% can be in the cis configuration • Proline isomerization is catalyzed by proline isomerases Protein Tertiary Structure • Tertiary structure refers to the overall spatial arrangement of atoms in a polypeptide chain or in a protein • One can distinguish two major classes – fibrous proteins ¤ typically insoluble; made from a single secondary structure – globular proteins ¤ water-soluble globular proteins ¤ lipid-soluble membraneous proteins Fibrous Proteins: From Structure to Function Function Structure Example Tough, rigid, hard (nails, horns) Cross-linked -helixes Rigid linker (S—S) -keratin Tensile strength, non-stretching (tendons, cartilage) Cross-linked triple-helixes Flexible linker (Lys-HyLys) Collagen Soft, flexible non-stretchy (egg sac, nest, web) Non-covalently held -sheets van der Waals interaction Silk fibroin Structure of Keratin in Hair Chemistry of Curly Hair Structure of Collagen • Collagen is an important constituent of connective tissue: tendons, cartilage, bones, cornea of the eye • Each collagen chain is a long Gly- and Pro-rich left-handed helix • Three collagen chains intertwine into a right-handed superhelical triple helix • The triple helix has higher tensile strength than a steel wire of equal cross section • Many triple-helixes assemble into a collagen fibril Collagen Fibrils 4-Hydroxyproline in Collagen • Forces the proline ring into a favorable pucker • Offer more hydrogen bonds between the three strands of collagen • The post-translational processing is catalyzed by prolyl hydroxylase and requires -ketoglutarate, molecular oxygen, and ascorbate (vitamin C) Silk Fibroin • Fibroin is the main protein in silk from moths and spiders • Antiparallel sheet structure • Small side chains (Ala and Gly) allow the close packing of sheets • Structure is stabilized by – hydrogen bonding within sheets – London dispersion interactions between sheets Spider Silk • Used for webs, egg sacks, and wrapping the prey • Extremely strong material – stronger than steel – can stretch a lot before breaking • A composite material – crystalline parts (fibroin-rich) – rubber-like stretchy parts Motifs (folds) Arrangements of several secondary structure elements Quaternary Structure • Quaternary structure is formed by spontaneous assembly of individual polypeptides into a larger functional cluster Structure of the Cro-DNA complex • 6Cro.pdb Albright, R. A. and B. W. Matthews (1998). "Crystal structure of lambda-Cro bound to a consensus operator at 3.0 A resolution." J Mol Biol 280(1): 137-51. Protein Stability and Folding •A protein’s function depends on its three-dimensional 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 Ribonuclease Refolding Experiment • Ribonuclease is a small protein that contains 8 cysteins linked via four disulfide bonds • Urea in the presence of 2mercaptoethanol fully denatures ribonuclease • When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed • The sequence alone determines the native conformation • Quite “simple” experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize • How Can Proteins Fold So Fast? Proteins fold to the lowest-energy fold in the microsecond to second time scales. How can they find the right fold so fast? • It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest energy one is found (Levinthal’s paradox) • Search for the minimum is not random because the direction toward the native structure is thermodynamically most favorable Chaperones Prevent Misfolding Chaperonins Facilitate Folding Protein Structure Methods: X-Ray Crystallography Steps needed: • Purify the protein • Crystallize the protein • Collect diffraction data • Calculate electron density • Fit residues into density Pros: • No size limits • Well-established Cons: • Difficult for membrane proteins • Cannot see hydrogens Proton NMR spectrum of a protein Amides Aromatics Alphas Aliphatics Methyls Structure Methods: Biomolecular NMR Steps needed: • • • • • Purify the protein Dissolve the protein Collect NMR data Assign NMR signals Calculate the structure Pros: • No need to crystallize the protein • Can see many hydrogens Cons: • Difficult for insoluble proteins • Works best with small proteins Chapter 4: Summary In this chapter, we learned about: • the two most important secondary structures: – helixes – sheets • how properties and function of fibrous proteins are related • how to determine three-dimensional structures of proteins • one of the largest unsolved puzzles in modern biochemistry: how proteins fold?