1 / 27

Folding

Folding. Anfinsen cooperativity time scales, speed range Levinthal paradox ensembles energy landscape; funnel chaperones thermodynamics, 15 kcal/mol denaturation: thermal, chemical 2-state vs. intermediates, phi-values contact order as a metric of "foldedness"

alexavier
Download Presentation

Folding

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Folding • Anfinsen • cooperativity • time scales, speed range • Levinthal paradox • ensembles • energy landscape; funnel • chaperones • thermodynamics, 15 kcal/mol • denaturation: thermal, chemical • 2-state vs. intermediates, phi-values • contact order as a metric of "foldedness" • lattice models (Shakhnovich, Dill, Skolnick)

  2. Folding • Anfinsen (1950’s) – showed reversibility of denaturation with urea for RNase A • amino acid sequence encodes struct; thermodynamic hypothesis • exception is chaperones (also role of disulfides, Pro isomerization) • folding is “cooperative” differential scanning calorimetry

  3. Time-scales for folding • cytochrome b562: 5 ms • lambda repressor: 0.67 ms • rat IFABP: 33 ms • CRABP 1: 24.5 sec • tryptophan synthase b2-subunit: 992 sec (396 aa)

  4. Kubelka et al (2004)

  5. Galzitskaya et al. (2003)

  6. Folding, Unfolding, and Re-folding • at equilibrium, proteins represent an ensemble, with some unfolded (constantly unfolding and refolding) • thermodynamic ensembles (Boltzmann distribution) • can measure with hydrogen-exchange (NMR) • even buried H’s exchange with solvent at some rate • reflects dynamic unfolding/refolding • overall folding rate const vs. kunfold and kfold • equilibrium shifted in direction of DG

  7. Thermodynamic vs. kinetic control? • do folded structures represent true global energy minimum, or just “kinetically accessible” local minima? • what causes slow folding: a high transition-state barrier, or just a large space to search?

  8. Levinthal Paradox • How can proteins fold in such a short time? • Number of degrees of freedom: • >2Nres (phi/psi angles), <3*10*Nres (atomic coords) • states: ~3N*3N? (backbone a/b/coil × side-chain rotamers) • how can this large space possibly be sampled to find the global minimum? • intermediates and cooperativity • collapse of hydrophobic core • formation of key secondary structures • folding “pathway” • off-pathway intermediates (local minima) can act as traps and slow-down the folding process

  9. energy landscape funnel • new view: not just one preferred path • many routes lead to min • hydrogen-exchange • natural/fast folding sequence have “minimally frustrated” energy landscapes

  10. Two-state folding • data must fit first-order kinetics • linearity of ln(kf) vs. [denaturant] • DG is same whether determined by kinetic vs. thermodynamic (equilibrium) methods • no intermediates (at least not well-defined) • what does the (transient) transition state look like? • molten globule (Ptitsyn): collapsed but not tightly-packed, rapidly fluctuating • stopped-flow hydrogen-exchange shows “native-like” secondary structure signatures (BPTI, a-lactalbumin) • bT – measure of where transition occurs along reaction coordinate: how “native-like”?

  11. Jackson and Fersht (1991) – chymotrypsin inhibitor 2 1. 2-state model supported by concordance of params between thermo. and kinetics 2. slope (mF and mU) correlates with difference in accessible surface area between U and F (Myers, Pace, and Scholtz, 1995) 3. if Ku=ku/kf and ku=kuH20+mf[GCl] and kf=kfH2O-mu[GCl], then m=mu+mf 3-state: barnase rates! re-folding (stopped flow) unfolding (fluorescence curve) equilibrium!

  12. van 't Hoff equation • thermal denaturation Gibbs-Helmholtz equation • Pace and Laurents (1989) • Method for determining DCp • - calorimeter (10% error) • DCp=d(DH)/dT from v’Hoff • extrapolate from DG • measured at different • denaturant concentrations balance between DS and DH

  13. Folding Pathway Intermediates • hard to trap (low populated) • non-linearity in chevrons in plots • due to switch of dominant transition state • intermediate CD spectra, hydrodynamic radius • barnase (Fersht, 2000, PNAS) • Sanchez and Keifhaber (2003) – multiple examples (conditions) • spectrin (Scott and Clarke, 2005) broad transition vs. sequential intermediate states?

  14. Lysozyme has both a fast a slow pathway (Keifhaber, 1995) – data fit better by a double-exponential (t1=50ms, t2=420ms) see also Jamin and Baldwin (1996). folding vs. unfolding rates as evidence for intermediates in apomyoglobin

  15. Valerie Daggett • molecular dynamics simulation of folding/unfolding • identification of order of sub-structure formation simulations of ubiquitin at 498 K and 298 K

  16. Off-pathway intermediates • BPTI – 3 native disulfide bridges, 14-38, 30-51, and 5-55 • other non-native bridges are formed during folding in an oxidizing environment • proper folding follows specific order of formation • making non-native disulfides forms “kinetic traps” • can block free thiols and analyze population; distribution suggests thermodymamically determined (equilibrium?) show picture of interconversion of intermediates...

  17. The Unfolded “State” • random coil? (hydrodynamic radius) • backbone, side-chains fully solvated (hydration) • effects of pH, urea...

  18. Contact Order • (Plaxco Simons & Baker, 1998) L = length of protein N = num of contact pairs (side-chain dist < 6A) DS = sequence separation 1HRC, CO=11.2 1TEN, CO=17.4 1UBQ, CO=15.1

  19. F-values • Fersht AR, Matouschek A, Serrano L. (1992) • a way of studying kinetics and folding intermediates via mutation • if you mutate a residue that is a critical (folded) part of an intermediate structure, you might destabilize it, increasing the barrier, and decreasing the rate of folding • if intermediate is structured and resembles native, then mutation will affect stability of each equally • it intermediate is unfolded, mutation will not affect stability of TS • examples: • Crespo, Simpson, and Searle (2006) – ubiquitin • Bulaj & Goldenberg (2001) - BPTI phi=0 no effect on TS phi=1 mutation affects TS

  20. Lattice Models • Sali, Shakhnovich, and Karplus (1994) • Monte Carlo sampling of configurations • simplified interactions: native contact=1, else 0 • modeling secondary structure • energy function: sum over all contacts • moves: swap to neighboring site, avoid self-intersection • Metropolis criterion: accept if DE<0 or with p>exp(-DE/kT) • study which factors determine whether a random sequence will fold (fast): • short-range vs. long-range contacts (contact order)? • size? secondary structure? hydrophobicity? • presence of a clearly-defined (deep) energy minimum

  21. order parameter for heterogeneity of ensemble (related to entropy) synthetic example of a compact folded polymer ends can’t move

  22. extensions • Dill, HP model: H and P atom types, 2D lattice • off-lattice models

  23. Kolinski, Godzik, Skolnick (1993) • ab initio folding? • Ca’s only, on-lattice model (1.7Å spacing) • side-chains modeled as spheres • statistical side-chain contact potential (eij) • non-directional H-bonds • 4-body side-chain interactions • cooperative coupling

  24. SICHO (Kolinski and Skolnick, 1998) • ab initio folding with a few (~20) restraints (e.g. NMR) • model side-chains centers only (no Ca’s) on-lattice • Monte Carlo moves – multiple groups of atoms • energy function: simplified geometry statistics, contact potentials

  25. Reduced-atom models • Go (1980) model (off-lattice) • Ca’s: beads on a string (bond dist/angle contraints) • good description in Hoang and Cieplak (2000). • energy function includes term for native contacts (springs) • application to mechanical unfolding of titin

  26. Mis-folding and Amyloid formation • aggregation vs. fibril formation • disease processes (20, Alzheimer’s, a-b) • DLS – dynamic light scattering • solid-state crystallography • kinetics (polymerization) • similarity between 2 global minima • “dual-basin” – mis-folded intermediate for GFP • Andrews et al (2008) • http://www.pnas.org/ content/105/34/12283 Dobson (1998)

More Related