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Fast Processes of a -Helix Folding

Fast Processes of a -Helix Folding. Martin Volk University of Liverpool. Workshop “Future Prospects for Macromolecular Dynamics on 4GLS” Jan 26, 2006. Overview. Experimental amide I vibration as sensor for secondary structure setup for nanosecond temperature jumps

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Fast Processes of a -Helix Folding

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  1. Fast Processes of a-Helix Folding Martin Volk University of Liverpool Workshop “Future Prospects for Macromolecular Dynamics on 4GLS” Jan 26, 2006

  2. Overview • Experimental • amide I vibration as sensor for secondary structure • setup for nanosecond temperature jumps • Results for short alanine-based peptides • correlation between side chain helix propensity and helix folding dynamics • direction dependence of helix folding • Opportunities for 4GLS

  3. Introduction -a-Helical Model Peptides Ac-AAKAAAAKAAAAKAAAAKAAGY-NH2  dynamic equilibrium between helical and disordered (random coil) structures : helix-coil relaxation time in alanine-based peptides: 150 - 500 ns

  4. Temperature Jump Technique a-helices melt at higher temperatures temperature jump observation of (un)folding dynamics

  5. Observation of Structural Changes Amide I vibration as sensor for secondary structure: 1655 cm-1 random coil 1630 cm-1a-helix peptide: Ac-AAAAA(AAARA)4A-NH2

  6. Temperature Jump Setup signal rise time: 14 ns

  7. Observation of Helix-Coil Relaxation Ac-AAAAA(AAARA)4A-NH2 20  23.5oC a-helix relaxation

  8. propagation constant s: s Side Chain Helix Propensity Ac-AAKAAAAKAAAAKAAAAKAAGY-NH2 residue helix propensity s A Ala 1.70 R Arg + 1.14 K Lys+ 1.00 E Glu- 0.54 Q Gln 0.62 Rohl et al., Prot. Sci. (1996), 5, 2623  effect of different side chains on helix dynamics?

  9. a-Helical Model Peptides AK: Ac-AAKAAAAKAAAAKAAAAKAAGY-NH2 AR: Ac-AARAAAARAAAARAAAARAAGY-NH2 AE: Ac-AAEAAAAEAAAAEAAAAEAAGY-NH2 AQ: Ac-AAQAAAAQAAAAQAAAAQAAGY-NH2 CD-spectra: helicity at 28oC AR 66% AK 60% AE 51% AQ 44% residue helix propensity s A Ala 1.70R Arg + 1.14 K Lys+ 1.00 E Glu- 0.54 Q Gln 0.62 Rohl et al., Prot. Sci. (1996), 5, 2623

  10. Results - Helix-Coil Dynamics of Different Peptides tRel / ns AR 184  2AK 194  4AE 147 2AQ 117 3 single exponential fits  different peptides have different helix-coil relaxation dynamics

  11. Results - Helix-Coil Dynamics of Different Peptides tRel / ns AR 184  2AK 194  4AE 147 2AQ 117 3 23  28oC  significant effect of different residues  max. relaxation time at > 60% helicity

  12. Correlation Helicity  Dynamics - Kinetic Modelling Two-state model kf/ ku = K kudecreased unfolding slowed down kfandkuaffected kfincreased folding accelerated increased helix stability:

  13. Correlation Helicity  Dynamics - Kinetic Modelling Two-state model  dynamics of folding and unfolding equally affected by different residues ?

  14. .... .... Correlation Helicity  Dynamics - Kinetic Modelling kinetic zipper model kp/ k-p = s kn/ k-n = ss Random coil Thompson, Eaton, Hofrichter, Biochemistry (1997), 36, 9200

  15. Correlation Helicity  Dynamics - Kinetic Modelling simulation results  dynamics of hydrogen bond breaking more affected by different residues than dynamics of hydrogen bond formation ?

  16. Isotope-Edited IR Spectroscopy Amide I Vibration 12C 13C 12C  13C : shift of nCO by 35 cm-1 12C 13Ca-helix 1630 cm-1 1595 cm-1random coil 1655 cm-1 1620 cm-1

  17. Isotope-Labelled Model Peptides 4A: AAAAKAAAAKAAAAKAAAAY-NH2 4AL1: AAAAKAAAAKAAAAKAAAAY-NH2 4AL2: AAAAKAAAAKAAAAKAAAAY-NH2 4AL3: AAAAKAAAAKAAAAKAAAAY-NH2 4AL4: AAAAKAAAAKAAAAKAAAAY-NH2 A: 12C, A: 13C Sean Decatur (Mount Holyoke College, MA)

  18. IR Spectra of Labelled Peptides 4A 12C 13Ca-helix 1630 cm-11595 cm-1r.c. 1655 cm-1 1620 cm-1 Decatur, Biopolymers 54 (2000) 180.  separate observation of a-helical structure in labelled peptide sections

  19. IR Spectra of Labelled Peptides peptide ends are “frayed”, middle sections are highly helical

  20. Results - Different Peptide Sections unlabelled peptide 4A + labelled peptides Temp. Jump: 4oC 9oC 12C 13Ca-helix 1630 cm-11595 cm-1r.c. 1655 cm-1 1620 cm-1  observation of helix-coil dynamics in labelled sections alone !

  21. Results - Different Peptide Sections labelled peptides Temp. Jump: 4oC 9oC time constant t: 4AL1 814ns4AL2 801ns4AL4 818ns 4AL3 730ns  helix-coil relaxation of third section is faster

  22. Results - Different Peptide Sections Helix-coil relaxation times of labelled sections  helix-coil relaxation of third section faster by 7-10% AAAAKAAAAKAAAAKAAAAY-NH2

  23. Section 2 Section 3 Section 4 Section 1 N-terminus C-terminus N-terminal versus C-terminal Dynamics peptide ends are “frayed”, middle sections are highly helical: N-terminalhelix end C-terminalhelix end dynamics of section 3 faster than section 2: a-helix folding/unfolding faster at the C-terminal helix end than at the N-terminal helix end

  24. C-terminus N-terminus N-terminal versus C-terminal Dynamics C-terminus: C=O free N-terminus: C=O hydrogen bonded  less steric restrictions at C-terminus  more flexibility at C-terminus ?

  25. N-terminal versus C-terminal Dynamics Distribution of j/y angles at helical ends (from protein data bank) Ho et al, Protein Sci.12 (2003) 2508. N-teminus C-terminus  more flexibility at C-terminal helix end

  26. N-terminus C-terminus N-terminal versus C-terminal Dynamics Observation: faster helix-folding/unfolding dynamics at C-terminus Hypothesis: Faster dynamics due to higher flexibility and easier solvent access?  allows optimisation of reaction pathway for H-bond formation?

  27. Summary • temperature jump experiments  observation of helix/coil dynamics with high time resolution • replacing hydrophilic residues in helical peptide has significant effect on helix-coil dynamics benchmark for MD-simulations ? effect of single replacement should be observable • isotopically labelled peptides allow the observation of local peptide dynamics a-helix folds more rapidly at the C-terminus than at the N-terminus

  28. Acknowledgements Angela Pozo Ramajo Dr. Ed Gooding (Swarthmore College, PA) Dr. Sean Decatur (Mount Holyoke College, MA) Dr. Sarah A. Petty Financial support EPSRC, University of Liverpool HHMI, Swarthmore College NSF + Dreyfus Foundation CCLRC LSF Laser Loan Pool

  29. How Will 4GLS Help? • UV-CD detection  Francois Haché http://www.cryst.bbk.ac.uk/cdweb/html/home.html

  30. How Will 4GLS Help? • 2D-IR 2D-IR spectrum of Trp-zipper:  David Klug, Kevin Kubarych http://rlbl.chem.upenn.edu/projects.htm

  31. How Will 4GLS Help? • THz detection bovine serum albumine in H2O: crystalline Ala3: p: parallel b-sheetap: anti-parallel b-sheet Xu et al., Prot. Sci. 15 (2006) 1175 http://physics.nist.gov/Divisions/Div844/facilities/thz/cwthz.html

  32. How Will 4GLS Help? • “single” shot measurements need good S/N  average 20,000 shots helical peptides very stable, folding fast + reversible  BUT: Other samples not reversible ? Other techniques not reversible ?

  33. How Will 4GLS Help? • “single” shot measurements Photochemical method: Disulfide trigger  Jon Waltho, Chris Hunter photodissociation triggers folding

  34. How Will 4GLS Help? • “single” shot measurements Photochemical method: pH jump photolysis of o-NBA Folding of a-helical poly-Glu after pH-jump (average of 10-20 shots) 625 ns Causgrove and Dyer, Chem. Phys. 323 (2006) 2

  35. Calibration – D2O Temperature-dependent FTIR spectra 1630 cm-1 1580 cm-1

  36. Fit Residuals

  37. Introduction -a-Helical Model Peptides Ac-AAKAAAAKAAAAKAAAAKAAGY-NH2 Host-guest studies  helix stability propagationN-capAla 1.70Arg + 1.14 1.00Lys+ 1.00 0.72Glu- 0.54 2.06Gln 0.62 0.12 Rohl et al., Prot. Sci. (1996), 5, 2623

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