CHAPTER 4 Proteins: Structure, Function, Folding

1. CHAPTER 4Proteins: Structure, Function, Folding • 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 Key topics:

2. 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

3. Protein Structures are compact Protein Sci. 2006 August; 15(8): 1829–1834. doi: 10.1110/ps.062305106.

4. 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

5. 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

7. 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°

9. 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 • 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 3

10. Ramachandran Plot

11. 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

13. 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

15. 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?

16. 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

17. 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

18.  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

19. Parallel and Antiparallel b Sheets • Parallel or antiparallel orientation of two chains within a sheet are possible • In parallelb sheets the H-bonded strands run in the same direction • In antiparallel b sheets the H-bonded strands run in opposite directions

22. 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

23. b Turns (Hairpins) • b-turns occur frequently whenever strands in b 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 2or glycine in position 3 are common in b-turns

24. 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

25. 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

26. Fibrous Proteins: From Structure to Function Function Structure Example Tough, rigid, Cross-linked a-helixes a-keratin hard (nails, horns) Rigid linker (S—S) Tensile strength, Cross-linked triple-helixes Collagen non-stretching Flexible linker (Lys-HyLys) (tendons, cartilage) Soft, flexible Non-covalently held b-sheets non-stretchy van der Waals interaction Silk fibroin (egg sac, nest, web)

27. Structure of -Keratin in Hair

28. Chemistry of Curly Hair

29. 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

30. Collagen Fibrils

31. 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 a-ketoglutarate, molecular oxygen, and ascorbate (vitamin C)

32. Silk Fibroin • Fibroin is the main protein in silk from moths and spiders • Antiparallel b 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

33. 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

34. Motifs (folds) Arrangements of several secondary structure elements

35. Quaternary Structure Quaternary structure is formed by spontaneous assembly of individual polypeptides into a larger functional cluster

36. 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.

37. 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

38. Ribonuclease Refolding Experiment • Ribonuclease is a small protein that contains 8 cysteins linked via four disulfide bonds • Urea in the presence of 2-mercaptoethanol 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

39. 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

40. Chaperones Prevent Misfolding

41. Chaperonins Facilitate Folding

43. 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

44. Proton NMR spectrum of a protein Amides Aromatics Alphas Aliphatics Methyls

45. 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

46. 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?