Exploring photolytic triggers of protein folding

1. Exploring photolytic triggers of protein folding Jon Waltho Department of Molecular Biology & Biotechnology The University of Sheffield Future Prospects for Macromolecular Dynamics on 4GLS Daresbury, 26-27 January 2007

2. Protein Denaturation and Refolding High Denaturant Folded State Unfolded State U Measured Parameter F F U Low Denaturant m-values correlate strongly with the degree of burial from solvent of hydrophobic residues Denaturant concentration Folding Unfolding Slope = "m value" Free Energy F U Denaturant concentration

3. Two-state Folding Funnels Smooth Golf-course Bumpy bowl (Dill & Chan 1997, Nature Struct. Biol. 4, 10-19)

4. Observation of Kinetic Intermediate States High Denaturant U I F F I Refolding U Low Denaturant Unfolding m-values correlate strongly with the degree of burial from solvent of hydrophobic residues Slope = "m value" Free Energy F I U Denaturant concentration

5. Free Energies of Folded and Kinetic Intermediate States relative to Unfolded States

6. Three-state Folding Funnels Stabilisation by a wide range of native contacts Stabilisation by a narrow range of native contacts Somewhere in-between (Dill & Chan 1997, Nature Struct. Biol. 4, 10-19)

7. 270 nm Photocleaving Restrained Non-Native States Crosslink incompatible with native fold Photocleavage Monitor refolding

8. Experimental Folding Models - Phosphoglycerate kinase (PGK) - CD2 d1 domain - Protein G

9. bsPGK (W290Y): Domain Folding W315 • Each domain forms an I-state independently • N-domain folds first • Slowest step is C-domain I-F transition (Parker et al., Biochemistry (1996) 35, 15740-15752)

10. Probing of Rare States using Amide Exchange kopen kint CH OH OD kclose Kinetic Intermediates CD2: Parker et al., Nature Struct. Biol. (1998) 5:194 Closed Form Open Form G kopen kclose . kint kobs = kopen kclose + kint F O Partially Folded States PrPc: Hosszu et al., Nature Struct. Biol. (1999) 6:740 C m Molten Globules bPrP: Jackson et al., Science (1999) 283:1935

11. Protection Factors in I state of intact PGK 2H,13C,15N-labelled PGK pH 7.5, 310K (Hosszu et al., Nature Struct. Biol. (1997) 4, 801-804) 1 174 371 394 (Reed et al., J. Mol. Biol. (2003) 330, 1189-1201)

12. Photocleavable crosslinked versions of N-PGK Mutations F17W, C18V E108C, R118C Choice of Attachment Site for Crosslinker - high effective molarity of cys residues - sufficient degree of fold disruption - cleaved residue tolerated in folded state

13. Protocol for Crosslinker Attachment Step 1: modification of both Cys Step 2: unfolding and exposure of the crosslinker Step 3: oxidation and refolding verification of product Electrospray MS

14. Testing the Authenticity of Crosslinkage Electrospray MS Crosslinked protein Cleavage of crosslink via disulphide reduction Verification of crosslink reduction via derivatisation (+ 250 Da = 2 x maleimide)

15. Characterisation of Crosslinked [C108,C118]-N-PGK Trp Fluorescence Circular Dichroism Reduced Crosslinked Crosslinked Reduced Crosslinked Crosslinked 330 nm 220 nm Reduced Reduced [GuHCl] [GuHCl]

16. 1H NMR of Crosslinked [C108,C118]-N-PGK Top: Crosslinked Middle: Reduced Crosslinked Bottom: Prior to Crosslinking 1.0 M GuHCl Crosslinked [E108C,R118C]-N-PGK is a molten globule in the absence of denaturant

17. Identification of molten globule states Folded Protein ppm Molten Globule • reduced resonance dispersion • resembles unfolded state ppm

18. Chemical shift changes in the denatured state 1H,15N HSQC crosspeaks versus GuHCl concentration Red = 1.3 M Black = 1.5 - 1.9M Blue = 2.0 - 4.0 M x-axis: 0.2 ppm 1H y-axis: 2.0 ppm 15N Resonance assignment: 20 kDa, 174 residues, pH 6.0, 25oC, 0.8 mM protein, 4M GuHCl

19. Comparison with SEC, CD and FL data N-PGK • High denaturant transition does not involve large scale collapse of the unfolded state: • Rate > 104s-1; mU-I1 < 2 M-1 • Lower denaturant transition does involve collapse: • Rate ~ 103-104 s-1; mU-I2 < 5 M-1 [GdnHCl] (mU-Ikin = 11 M-1; mU-F = 16 M-1)

20. Conformations in the denatured state - Denatured state helices are longer than those in the native state - RDCs indicate parallel or antiparallel - 4/5 helices in low denaturant concentration, denatured state Ikin F U I2 (Reed et al., J. Mol. Biol. (2006) 357, 365-372)

21. Experimental Folding Models - Phosphoglycerate kinase (PGK) - CD2 d1 domain - Protein G

22. CD2 Domain I: Folding behaviour Kinetics of folding/unfolding at pH 5, 7 and 9 kF-I by NMR KF/I = 3.4x104 KI/U = 8.0x101 Ig-V fold kI-F = 6.7 s-1 kF-I = 2.0x10-4 s-1 98 residues No disulphides

23. CD2: Competition vs Equilibrium Amide Exchange U 2 Competition log10PI 0 I U I 4 Equilibrium log10PF 2 0 F 20 40 60 80 -Values extrapolated to EX2 limit -Filled Bars = EX1 limit at pH 9.0 (Parker et al., Nature Struct. Biol. (1998) 5:194-198)

24. CD2 Domain I: Comparison of equilibrium and competition methods White: Exchange controlled by kF-I in equilibrium experiment B E E F C B F C C E F Red: Exchange protected in competition experiment B Most Ig folds have a disulphide bond between strands B and F

25. CD2 Domain I: Double Cysteine Mutants 13 Double thiol mutations distributed throughout the fold G13C,K66C E33C,L38C N15C,K64C T75C,D94C L58C,D62C R31C,E41C T79C,N90C E29C,K43C Y81C,R87C M23C,N60C Also Core Mutants: [I18C,V78C]; [A40C,L50C]; [I57C,L63C] (Mason et al., Biochemistry (2002) 41:12093-12099)

26. Crosslinkage of [C29,C43]-CD2.d1 E29C,K43C E29C,K43C [C29,C43] disulphide links strand C and C’

27. Characterisation of Crosslinked [C29,C43]-CD2.d1 Circular Dichroism Denaturant Dependence Crosslinked Non-crosslinked [GuHCl] M Wavelength (nm) - Crosslinked [C29,C43]-CD2.d1 is a beta-rich molten globule - A beta-rich molten globule component is apparent in the denatured state of the non-crosslinked protein

28. Experimental Folding Models - Phosphoglycerate kinase (PGK) - CD2 d1 domain - Protein G

29. Protein G: Double Cysteine Mutants 2-State Folding Behaviour T25C,N37C T25C,D46C Current Double Cysteine Mutants - [C25,C37] = Distortion of Helix - [C25,C46] = Separation of Helix and Sheet

30. Testing the Authenticity of Crosslinkage MALDI MS 7736 7738 (Crosslinked [C25,C46]-Protein G + NEM) Crosslinked [C25,C46]-Protein G 7739 Cleavage of crosslink via disulphide reduction 7988 Verification of crosslink reduction via derivatisation (+ 250 Da = 2 x NEM)

31. 1H,15N-HSQC NMR shows residual structure in crosslinked [C25,C46]-Protein G 1H,15N-HSQC in water (Mixture of crosslinked and non-crosslinked species) 15N (ppm) Blue peaks = Crosslinked Red peaks = Non crosslinked 1H (ppm) - Crosslinked [C25,C46]-Protein G is a molten globule in water - Crosslinked molten globule unfolds before reduced folded form

32. 270 nm Photolysis: Choice of Restraining Agent Aminothiotyrosine Amino aryl disulphide derivatives based on the work of Volk, Hochstrasser and DeGrado (Lu et al.,J. Am. Chem. Soc. 1997, 119, 7173-7180; Volk et al.,J. Phys. Chem. B 1997, 101, 8607-8616)

33. Photocleavage of model crosslinkers DOD time (ps) Cleavage: 100 fs pulse (260 nm; 100 nJ) Detection: Absorbance (520 nm) (Milanesi et al. 2004, Chemistry10, 1075-1710)

34. Photocleavage of [C108,C118]-N-PGK crosslinked with p-maleimidothiophenyl groups

35. Summary The next phase for photocleavage - Higher sensitivity detection of folding transitions - Lower recombination of cleaved products Photocleavable crosslinks can be readily introduced PGK Large a/b fold with stable Ikin state and equilibrium MG state CD2 Medium sized all b fold with metastable Ikin state Protein G Small a/b protein that folds without a stable Ikin state

36. Acknowledgements University of Sheffield Lilia Milanesi Dominic Sloane Clare Jelinska Chris Hunter Jeremy Craven Rosie Staniforth Andrea Hounslow Tooba Alizebah Laszlo Hosszu Martin Parker Matthew Cliff Michelle Reed Andrew Splevins Karl Syson University of Bristol Tony Clarke University of Leeds Godfrey Beddard Gavin Reid Daniel Shaw University of Liverpool Martin Volk Sagarika Dev Daresbury Laboratory Dave Clarke Gareth Jones