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Protein Folding

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Protein Folding

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    1. Protein Folding Thermodynamics vs. kinetics the “Levinthal paradox” In vitro forces In vivo chaperones Reading: Whitford, ch. 11 Lehninger ch. 4.4 B&T, ch. 6 Advanced Dill and Chan, Nat. Struc. Biol., 1997, 4, pp.10-19 Dinner et al., TIBS, 2000, 25, p.331

    2. Protein Folding Related Diseases

    3. Review of Thermodynamics DG Gibbs free energy DH enthalpy DS entropy Cp heat capacity

    4. Some Useful Physical Constants RT 2.48 kJ/mol eo 8.854×10-12 C2 N-1 m-2 is the vacuum permitivity er dielectric constant = scaled vacuum permitivity Water 78.5 Ethanol 24.3 Benzene 2.27 Hydrophobicity the transfer of a non-polar solute to an aqueous solvent accompanied by a large change in Cp

    5. Characteristics of a folded protein A well defined, generally hydrophobic core A generally polar, charged surface A unique folding pattern under defined conditions

    6. Thermodynamics of Protein Folding

    7. The Experiments of Anfinsen

    9. The forces that drive folding

    10. Hydrophobic Effects Definition: Transfer of non-polar solutes to an aqueous solution Primary driving force of protein folding! Evidence 3D structures – cores are mostly hydrophobic residues DGfold and DGtransfer (the energy required to transfer a hydrophobic mol. from an organic solvent to water) similar dependence on T Protein stability follows Hofmeister series (SO42-,CH3COO-,Cl-,Br-,ClO4-,CNS-) benzene is increasingly soluble in these ions Mutation studies Computer simulations

    11. Hydrophobic Effects-continued At room temp the “hydrophobic effect” is entropic water molecules form ordered structures around nonpolar compounds Hydrophobic residues collapse in to exclude water Additional forces can then stabilize (vdw, h-bond,intrinsic properties) Hydrophobic effect is dependent on temperature (unstable at high AND low temp).

    13. Electrostatic Contributions Fi is the attractive/repulsive potential energy between the charges zi is the unit difference in charge between the 2 ions e is the charge of the e- (1.602 × 10-19 C) eo is the dielectric constant r is the distance between the 2 ions Sensitive to pH and ion concentrations pH determines total charge (pI) Ionic strength determines effective range of interactions Ion pairs contribute 1-3 kcal/mol (on surface) Ion pairs generally destabilizing if buried (cost up to 19 kcal/mol/ion to completely bury Ion pairs contribute ~5-15 kcal/mol per 150 aa’s

    14. Hydrogen Bonds ~90% of CO and NH groups H-bonded in a folded protein, but nearly 100% are H-bonded in an unfolded protein in water. What are the differences? Hydrogen bonds contribute 2-10 kcal/mol Destabilizing by themselves (transfer of polar groups from high to low dielectric medium If driven by other forces (e.g. hydrophobic collapse) favour “internal organization”

    15. Opposing Effects Entropy! A folded protein has many fewer conformational states than an unfolded one!

    16. How many conformations are there in the Native state?

    18. Kinetics of Protein Folding “diffusion model” Many parallel pathways operating independently (water down the mountain) Steps are characterized by ensembles instead of unique conformations Modeled by simple chains embedded in a lattice (statistical mechanics)

    19. The Unfolded State

    20. Energetics and Kinetics

    21. Energetics and Kinetics

    22. Energetics and Kinetics

    23. Energetics and Kinetics

    24. Structure of the Transition State

    25. Solution to the Levinthal Paradox

    26. Energetics and Kinetics

    27. More Complex Proteins = More Complex Folding: Lysozyme

    29. The Folding of a Helix Folding@home

    30. Something a Little Bigger

    31. Real World Example

    32. Biologically Assisted Folding: Chaperones

    33. Properties of Chaperones Molecular chaperones interact with unfolded or partially folded protein subunits, e.g. nascent chains emerging from the ribosome, or extended chains being translocated across subcellular membranes. They stabilize non-native conformation and facilitate correct folding of protein subunits. They do not interact with native proteins, nor do they form part of the final folded structures. They also do not bind natively unfolded proteins. Some chaperones are non-specific, and interact with a wide variety of polypeptide chains, but others are restricted to specific targets. They often couple ATP binding/hydrolysis to the folding process. Essential for viability, their expression is often increased by cellular stress. Main role: They prevent inappropriate association or aggregation of exposed hydrophobic surfaces and direct their substrates into productive folding, transport or degradation pathways. Two Classes Constitutive – the chaperonins (GroEL/ES) Induced by stress – the HSP

    34. Mechanism of Action of Chaperones

    35. The GroEL folding machine

    36. Bacterial Folding Complexes

    37. The conformational cycle of GroEL/ES

    39. GroEL-ES in action

    40. Take Home Lessons Protein folding is governed by thermodynamics, however the time it takes is controlled by kinetic limitations. DGfold is usually a small negative number. However, it is made up of large, opposing numbers (entropy and enthalpy) which nearly balance out. That’s why it is exceptionally difficult to predict DGfold. The hydrophobic effect is the most important force driving folding. This is primarily an enthalpic phenomenon. The kinetics of protein folding can be fast because an individual molecule doesn’t have to sample a large number of conformations. Cells have evolved a variety of methods to assist protein folding and protect against misfolding. This (un)folding machinery is usually ATP dependent. The “folded” state of a protein actually consists of a number of rapidly interconverting, similar conformers.

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