Protein folding. Today s Outline

Transcription

1 Protein folding Today s Outline Review of previous sessions Thermodynamics of folding and unfolding Determinants of folding Techniques for measuring folding The folding process The folding problem: Prediction of 3-D structure 1

2 Forces that determine protein folding Intrinsic propensities amino acid conformations and permitted torsion angles Hydrophobic interactions Van der Waals interactions Dipole-dipole interactions Ionic interactions Hydrogen bonds W Thermodynamic considerations G = H T S H S ln K ~ bonding W = R ln( ) W o = # ofways G = RT Enthalpic contributions from non-covalent interactions Entropy from conformations, no. of ways possible Note: water, solvent also must be considered: hydration shells around solutes 2

3 Thermodynamic considerations Non-covalent interactions vs. some typical covalent bond energies Adapted from KE Van Holde, WC Johnson, PS Ho (1998) Principles of Physical Biochemistry, Prentice-Hall, p. 10. Thermodynamic considerations Type of Interaction Charge-charge Charge-dipole Dipole-dipole Charge-induced dipole Dispersion Steric repulsion Distance Relationship 1/r (Coulomb s) 1/r 2 1/r 3 1/r 4 1/r 6 1/r 12 And don t forget the dielectric constant! Water = ~80 (78.5) Κε 0 interior can be ~3.5 (about 1-20) Κε 0. 3

4 Water Structure Left, water structure; right, water cage around a methane molecule; center, clathrate of 24x14 water tetrahedra Sep 3. Red clathrate: Martin Chaplin (2007) Water Structure and Science, Updated 1 July Packing Jigsaw puzzle vs. beads in a jar 4

5 Summary of forces that fold globular proteins All residues must have stereochemically allowed conformations. Main chain and side chain atoms Steric collisions raise the energy and decrease stability Buried polar atoms must be hydrogen bonded to other buried polar atoms. After all, in the denatured, open conformation, these residues can interact with water Enough hydrophibic surface must be buried, and the interior must be densely packed, to provide thermodynamic stability After all, hydrophobic surfaces require ordered water! Today s Outline Review of previous sessions Thermodynamics of folding and unfolding Determinants of folding Techniques for measuring folding The folding process The folding problem: Prediction of 3-D structure 5

6 An intro to CD Circular Dichroism Principle of CD Incident linearly polarized light, where points 1-5 correspond to equal increasing time intervals. (a) Incident linearly polarized light. (b) Elliptically polarized light after passing through an optically active sample. (c) Resolution of linearly polarized light into left and right handed circularly polarized components. (d) Effect of optically active sample on the two circularly polarized components, where sum in (d) is equal to result in (b). Figure 8.1. Biophysical Chemistry Part II. Techniques for the study of biological structure and function. CR Cantor & PR Schimmel New York: WH Freeman, p Figure 8.1. Biophysical Chemistry Part II. Techniques for the study of biological structure and function. CR Cantor & PR Schimmel New York: WH Freeman, p

7 Measuring CD signals φ = opticalrotation 180l( nl n φ = λ θ = ellipticity θ = 2.303( A A l = samplelength A = absorbance L R ) deg R 180 ) deg 4π [ φ] = residuemolarrotation 100φ [ φ] = C l residue [ θ ] = residuemolarellipticity 100θ [ θ ] = C l residue CD Spectra for alpha-helix, beta-sheet, and random coil conformations VP Saxena & DB Wetlaufer (1971) Proc. Natl. Acad. Sci. USA 66:969. In: CR Cantor & PR Schimmel New York: WH Freeman, p. Figure 8-9, p

8 Measuring the thermodynamics of folding Guanidinium chloride unfolding of phosphoglycerate kinase using fluoresence (filled circles) and CD (open circles). A. Plot of raw data and spectral baselines for folded and unfolded. B. Unfolding curve generated from A. Adapted from H. Nojima et al. (1977) J. Mol. Biol. 116: In: TE Creighton (1993) Proteins: Structures and Molecular Properties, 2/E, Fig. 7.11, p Unfolding by heat Unfolding of RNAse A in HCl-KCl ph 2.1 and ionic strength. Measured by viscosity (open squares), optical rotation at 365 nm (open circles), UV A 287 (open triangles). Closed triangles from 2 nd melting after cooling. Adapted from A Ginsburg & WR Carroll (1965) Biochemistry 4: In: TE Creighton (1993) Proteins: Structures and Molecular Properties, 2/E, Fig. 7.11, p

9 Unfolding in acid Unfolding of staphylococcal nuclease A. Measured by viscosity (squares), circular dichroism at 220 nm (triangles). Open symbols- acidification; solid symbols- on raising ph. Adapted from CB Anfinsen(1972) Biochem. J. 128: In: TE Creighton (1993) Proteins: Structures and Molecular Properties, 2/E, Fig. 7.11, p What curves suggest Folding is a two-state process (for single-domain proteins). Proteins are either unfolded or folded, with minimal partially folded intermediates. K G G m eq = [ native] = [ unfolded] folding folding slope = G N = G G U H2O folding = RT ln K eq + m[ denaturant] 9

10 Today s Outline Review of previous sessions Thermodynamics of folding and unfolding Determinants of folding Techniques for measuring folding The folding process The folding problem: Prediction of 3-D structure The Classic Experiment Anfinsen s RNAse experiment 10

11 Bovine RNAse Sequence RNAse A disulfide bonds C40-C95: left: upper left; right: leftmost C84-C26: left: lower left; right: center left C65-C72: left: upper right; right: upper right C110-C58: left: lower right; right: lower right 11

12 Ribonuclease disulfides Ribonuclease folding 12

13 Anfinsen ribonuclease experiment Today s Outline Review of previous sessions Thermodynamics of folding and unfolding Determinants of folding Techniques for measuring folding The folding process The folding problem Prediction of 3-D structure 13

14 Levinthal s Paradox A protein cannot try all possible conformations because there is not enough time There must a pathway or process Mystery: Proteins are marginally stable Denatured state is heterogeneous Folding funnel 14

15 Folding models Folding general in the cell 15

16 Chaperones MCB animation 16

17 Today s Outline Review of previous sessions Thermodynamics of folding and unfolding Determinants of folding Techniques for measuring folding The folding process The folding problem Prediction of 3-D structure The Protein Folding Problem Given a protein sequence, find the 3-D structure The inverse protein folding problem: Given a protein 3-D structure, find the sequence Note: Many sequences are consistent with one general structure 17

18 Overview of Methods for 3-D Structure Prediction from Sequence Secondary structure prediction without full 3-D structure Homology modeling: prediction based on the known structures of one or more related proteins (at best comparable to a low-resolution experimental structure) Fold recognition: given a library of known structures, which of them shares a folding pattern with the query sequence of unknown 3-D structure, or if no fold matches the query sequence (analogous to a multiple-choice exam) Prediction of novel folds (a priori or knowledgebased), typically main chain only (analogous to an essay exam) Structural alignment 2 di rmsdeviation = n d = distance between ith pair of i n = number of points So: match corresponding atoms Find the distances Minimize the differences atoms 18

19 Prediction of function Sequence Structure Function But as proteins evolve they may Retain function and specificity Retain function but alter specificity Change to a related function, or a similar function in a different metabolic context Change to a completely unrelated function 19