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Proteins: The Unfolding Story

Proteins: The Unfolding Story. Protein Folding Problem. 1931 - Hsien Wu 1936 - Mirsky & Pauling 1950s - Anfinsen. Protein Folding - spontaneous acquisition of native conformation under physiological conditions. Who needs models?. Human Cognition. Seeing is believing Seeing is deceiving.

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Proteins: The Unfolding Story

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  1. Proteins:The Unfolding Story

  2. Protein Folding Problem 1931 - Hsien Wu 1936 - Mirsky & Pauling 1950s - Anfinsen Protein Folding - spontaneous acquisition of native conformation under physiological conditions

  3. Who needs models? Human Cognition Seeing is believing Seeing is deceiving

  4. Preview U N The Levinthal Paradox Sterics The Folding Reaction Re-equilibration

  5. Protein folding is a thermodynamic problem The problem has been with us for ~70 years What’s missing?

  6. The Folding Reaction - what we know Ginsburg and Carroll (1965)

  7. The Folding Reaction - what we don’t know U N ?

  8. Random Coil Model •No strongly preferred backbone conformations •Energy differences among backbone conformations ~kT •Statistical coil - chain configuration only becomes Gaussian for infinite chains. All real polymers are statistical coils.

  9. The Freely Jointed Chain Radius of gyration, Rg, measures coil dimensions Mean-square end-to-end distance grows as root

  10. Native state The Central Thermodynamic Question in Protein Folding How can a polypeptide chain overcome conformational entropy and fold to its native state?

  11. The Levinthal Paradox If each f,y pair can adopt either of two values, then a chain of 100 residues has 2100 = 1030 accessible conformers. A dilute protein solution contains ~1015 molecules.

  12. Why haven’t hard-sphere sterics played a more significant role in protein folding studies? Ramachandran, Ramakrishnan, Sasisekharan (1963) Influence of sterics does not extend beyond the dipeptide

  13. If each f,y pair can adopt one of two values, then a chain of 100 residues has 2100 = 1030 accessible conformers. Flory isolated-pair hypothesis, the simplifying assumption that each f,y pair is sterically independent. The Unfolded State How large is conformational space?

  14. “The Flory isolated-pair hypothesis is not valid for polypeptide chains - implications for protein folding” Pappu, Srinivasan & Rose PNAS (2000) 97:12565-12570. “A simple model for poly-proline II structure in unfolded states of alanine-based peptides” Pappu & Rose Protein Sci. (2002) 11:2437-2455. Rohit V. Pappu

  15. How to count: tiling f,y-space into mesostates Revisiting Ramachandran, Ramakrishnan & Sasisekharan

  16. If the Flory Isolated Pair hypothesis is valid, then the overall acceptance ratio for longer strings will be the product of the individual dipeptide acceptance ratios. How to count: tiling f,y-space into mesostates Generate 50,000 random dipeptide conformers within each mesostate, and capture the Acceptance Ratio. The acceptance ratio is the fraction of conformers that is clash free. Each mesostate has an acceptance ratio that ranges from 0 to 1. But it’s not!

  17. The isolated-pair hypothesis does not hold for polypeptides Exhaustive counting (in allowed regions of allowed mesostates) No. expected = No. generated x P acceptance ratios + 100 random extended mesostate strings * 100 random contracted mesostate strings

  18. The Unfolded State How shall I conform thee, let us count the ways. Polyalanine = pure backbone Sterics + hydrogen bond Two atoms can’t be in the same place at the same time.

  19. For energy = e: Weight ith string= giallowede[-bui(e)]/Z[14N] For e = 0 (sterics only): Weight ith string= giallowed/Z[14N] With gi’s obtained from acceptance ratios or sampling, depending on the string. How large is conformational space? Let us count the ways. Exhaustive counting of 14N strings (N=7): 7.5x106 strings, 5x108 conformers/string Then Sort

  20. A 7-residue polyAla chain is 2-state Tug of war between H-bond enthalpy and conformational entropy

  21. Energetic preference for particular structure? 2) Steric clash in hybrids? Why is the folding of a short polyalanine chain largely two-state? Specifically, why are a/b hybrid conformers depleted in this population? Yes - e.g, polyproline II Yes

  22. Thermodynamics vs. Structure If structurally distinct confomers can inter-convert via spontaneous fluctuation, they do not differ thermodynamically. CV = <E>2 - <E2>/RT2 When are two conformers distinguishable: structurally? thermodynamically? Investigator Space ® Protein Space Spontaneous fluctuations are free

  23. Do fluctuations occur about preferred basins? Identify the basins: •Without explicit solvent •With explicit solvent

  24. Inverse Power PotentialU = SiSj<i(sij/rij)nS = hard sphere contact distancer = interatomic separation Pappu & Rose (2002) Protein Sci. 11:2437-2455 Without explicit solvent Minimize density of chain around itself: mimic good solventand maximize wiggle room, i.e. chain entropy Hard sphere ® Soft sphere repulsive potential

  25. Without explicit solvent Polyalanine 7-mer. Global minimum is left-handed polyproline II helix

  26. Solvation free energy of a 12-residue polyAla peptide posed in 4 conformers DA (kcal/mol/res) Helix -2.0±0.3PII -4.7±0.3||b -3.9±0.3anti||b -4.0±0.3 Mezei et al (2004) Proteins 55: 502-507 With explicit solvent Prof. Mihaly Mezei Dept. Physiology & Biophysics Mt. Sinai School of Medicinehttp://inka.mssm.edu/~mezei Grand canonical ensemble Monte Carlo simulations in TIP3P water

  27. With explicit solvent b-strand Polyproline II b-strand with water bridges Mezei et al (2004) Proteins 55: 502-507

  28. Experimental confirmation Polyproline II structure in a sequence of seven alanine residues. Shi et al (2002) PNAS 99: 9190-9195. At 2° C, the chain is ~90% PII. At 55°, the chain is ~80% PII, ~10% strand. Large enthalpy PII®strand. Connecting thermodynamics and structure A few basins account for most of the thermodynamic population. The basins are highly degenerate. Most of the chain is within a room-temperature fluctuation of left-handed polyproline II helix.

  29. Energetic preference for particular structure? 2) Steric clash in hybrids? Why is the folding of a short polyalanine chain largely two-state? Specifically, why are a/b hybrid conformers depleted in this population? Yes - e.g, polyproline II Yes

  30. But what kinds of clashes? Are there steric restrictions beyond the alanyl dipeptide? Systematic local steric clashes winnow the number of accessible chain conformations •Depletion of mixed conformers serves to stiffen the chain.•Reduction in the number of conformers also promotes structure in the unfolded state.

  31. Steric restrictions beyond the alanyl dipeptide An a-helix cannot be followed by a contiguous b-strand Fitzkee & Rose Protein Science (2004) 13: 633-639.

  32. Definition of a, b, PII

  33. PDB select has 37,563 occurrences of a,a,a,~a but only 7 of these are a,a,a,b Two Tests 1. Examine the database 2. Hard sphere simulations

  34. Two Tests 2. Hard sphere simulations a4-*-a4

  35. Consequences of eliminating a-b hybrids •Winnows the size of conformational space.•Limits the number of protein domains.•Simplifies molecular recognition.•Suggests the possiblity of additional steric restrictions.

  36. U N The Levinthal paradox The size of accessible conformational space is much smaller than previously believed The folding reaction Unfolded population is more folded than we thought -- repopulation under changed solvent/temperature conditions Conclusions

  37. Rohit PappuWashington Univ.-St. Louis Raj SrinivasanTCS - Hyderabad, India Teresa PrzytyckaNIH Recent lab members

  38. Nick Fitzkee Pat Fleming Haipeng Gong Venk Murthy Nick Panasik Current lab members

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