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PROTEIN PHYSICS LECTURE 20

PROTEIN PHYSICS LECTURE 20. Protein Structures : Kinetic Aspects (2). Protein folding WITHOUT intermediates Nucleation of protein folding Folding nucleus Two- and multi-state folding.

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PROTEIN PHYSICS LECTURE 20

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  1. PROTEIN PHYSICS LECTURE 20 Protein Structures: Kinetic Aspects (2) • Protein folding WITHOUT intermediates • Nucleation of protein folding • Folding nucleus • Two- and multi-state folding

  2. … the main question as to how the protein chain can find its native structure among zillions of alternatives remained unanswered. A progress in the understanding was achieved when the studies involved small proteins (of 50 - 100 residues). Many of them are “two-state folders”: they fold in vitro without any observable (accumulating) intermediates, and have only two observable states: the native fold and the denatured coil.

  3. “Two-state” folding:without any observable (accumulating in experiment) intermediates NO LAG The “two-state folders” fold rapidly: not only much faster than larger proteins (not a surprise), but as fast as small proteins having folding intermediates (that were expected to accelerate folding…)

  4. However, what to study in the “two-state” folding where there are no intermediates to single out and investigate? Answer: just here one has the best opportunity to study the transition state, the bottleneck of folding. “detailed balance”: the same pathways for DN and ND “detailed balance”: the same pathways for DN and ND

  5. “Chevron plots”: Reversible folding and unfolding even at mid-transition, where kDN = kND N ===============N’ ===D’===============D   N D (a) (b)

  6. Folding nucleus: Directed mutations can show what residues belong to the native-like part of the transition state (“nucleus”), and what residues do not (Fersht, 1989) -ln(kN) folding unfolding inside V88A -ln(kN/kU) in- side L30A out- side folding unfolding outside

  7. “Chevron plots”:  equal slopes: evidence that energy and entropy (a), as well as the chain-to-solvent interface (b) of the transition state are between the ones of the native and the denatured states (when a vicinity of mid-transition is considered). (a) (b)

  8. Is given residue in nucleus or not? Mutate residue to Ala or Gly; estimate:  = ln(kN) / ln(kN/kU) If  1, residue is in nucleus; If  0, residue is not in the nucleus; If  0.5, residue is at the nucleus surface. =0.25  1  0 >>1 NEVER NEVER

  9. Folding nucleus in CheY protein (Lopez-Hernandes & Serrano, 1996) • In nucleus  > 0.3 • Outside  < 0.3  “difficult” residues:  = ?, since ln(kU)0 ln(kN)0 ln(kU/kN)0 Folding nucleus is often shifted to some side of protein globule and does not coincide with its hydrophobic core

  10. Significant modification of protein chain, such as its circular permutation, sometimes changes position of the folding nucleus, though the overall structure of the protein is preserved permutant

  11. Difference: Far from mid-transition

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