1. Using two recently-developed molecular dynamics protocols for protein folding Timothy H. Click Department of Chemistry and Biochemistry University of Oklahoma Norman, Oklahoma

2. 2Outline  Introduction to MD protocols  Previous work  Simulations of tryptophan zipper 2  Simulation of Streptococcal protein G B1 domain (residues 41-56)  Conclusions  Future directions  Acknowledgements

3. 3 Protein geometry optimization Dill, K.A.; Chan, H.S. Nat. Struct. Biol., 1997, 4, 10-19.

4. 4 1 st MD protocol — DIVE  Disrupted Velocity (DIVE) search protocol  Velocity reassignment of coordinate histories  Magnitude rescaling — energy perturbation  Direction changes  Reassignment every n steps (defined by user)  Heating and cooling cycles  Conformations sampled near absolute zero  Overall, protocol disrupts equilibrium  Energy barriers overcome or circumvented  Several potential energy minima sampled

5. 5 How conformations are selected βlow to DIP βlow2 to DIP β βlow

6. 6 2 nd MD protocol — DIP  Divergent Path (DIP) search strategy  Coordinate histories at same constant temperature  Simulations involve multiple coordinate histories  Individual coordinate histories randomly assigned initial velocities  Velocities can be altered allowing for different conditions  Constant temperatures maintained by rescaling velocity magnitudes  Broader sampling of potential energy surface allowed

7. 7 DIP simulation

8. 8 DIP simulation (cont’d) = -624.22 ± 8.27 kcal/mol = 1.6 ± 0.5 Å = -632.42 ± 8.44 kcal/mol = 1.8 ± 0.2 Å = -587.16 ± 9.50 kcal/mol = 13.2 ± 0.5 Å = -633.94 ± 7.87 kcal/mol = 1.5 ± 0.2 Å = -592.02 ± 8.38 kcal/mol = 12.5 ± 0.5 Å = -600.29 ± 13.05 kcal/mol = 11.3 ± 0.7 Å nmr

9. 9 Protocol procedures  Modified Amber force field (Okur,A.; Strockbine, B.; Hornak, V.; Simmerling, C., J. Comput. Chem., 2003, 21)  Constraints on atoms covalently bonded to hydrogen  Implicit solvent  2 fs time step  4,000,000 steps  Velocity disruption every 20,000 steps (DIVE)  T = 300 ± 20 K (DIP)  6 independent coordinate histories/simulation

10. 10 Previous work with α-helices  Zunnan Huang  13-residue polyalanine  Trp-cage (α-helix and 3 10 -helix  Huang and Zhanyong Guo  Peptide F  Timothy H. Click  C-peptide of ribonuclease A (residues 1-13)

11. 11 Tryptophan zipper 2 (trpzip2)  De novo 12-residue polypeptide  Sequence (S 1 WTWENGKWTWK 12 -NH2)  PDB code 1LE1 (20 NMR models)  Stable β-sheet in aqueous solution by cross-stranded pairs of four tryptophans  Simulations completed by other groups 1 Cochran, A.G.; Skelton, N.J.; Starovasnik, M.A. P. Natl. Acad. Sci. USA, 2001, 98, 5578-5583.

12. 12 Trpzip2 DIVE Results extlow E = -496.59 kcal/mol RMSD 6.9 Ẳ βlow E = -494.05 kcal/mol RMSD 5.0 Ẳ αlow E = -498.22 kcal/mol RMSD 6.1 Ẳ β* E = -489.44 kcal/mol RMSD 0.9 Ẳ extlow2 E = -499.23 kcal/mol RMSD 7.0 Ẳ βlow2 E = -497.06 kcal/mol RMSD 5.7 Ẳ αlow2 E = -498.17 kcal/mol RMSD 6.1 Ẳ

13. 13 Trpzip2 DIP Results ext = -360.82 ± 7.66 kcal/mol 6.5 ± 0.8 Ẳ β = -381.65 ± 4.55 kcal/mol 0.9 ± 0.1 Ẳ α = -369.79 ± 9.01 kcal/mol 6.7 ± 0.4 Ẳ extlow = -375.47 ± 6.28 kcal/mol 7.5 ± 0.1 Ẳ β low = -374.14 ± 7.18 kcal/mol 7.0 ± 0.3 Ẳ α low = -368.77 ± 7.38 kcal/mol 6.1 ± 0.1 Ẳ extlow2 = -381.44 ± 5.74 kcal/mol 7.4 ± 0.1 Ẳ β low2 = -378.75 ± 6.04 kcal/mol 7.0 ± 0.3 Ẳ α low2 = -368.93 ± 7.54 kcal/mol 6.2 ± 0.3 Ẳ β* = -381.65 ± 5.04 kcal/mol 0.8 ± 0.1 Ẳ

14. 14 Trpzip2 Summary  PES rough at low temperatures  β-hairpin challenging secondary structure  β-hairpin as relative global PE conformation  α-helices metastable conformation

15. 15 B1 domain of Streptococcal protein G  Natural β-hairpin stable in aqueous solution.  Sequence (G 41 EWTYDDATKTFTVTE 56 )  PDB 2GB1 (x-ray crystal structure)  Stabilization factors  Hydrophobic core  Terminal salt bridge  Several simulations 2 Gronenborn, A. M.; Filpula, D. R.; Essig, N. Z.; Achari, A.; Whitlow, M.; Wingfield, P. T.; Clore, G. M. Science, 1991, 253, 657-661.

16. 16 Protein G DIVE results extlow E = -784.40 kcal/mol RMSD 7.4 Ẳ βlow E = -774.96 kcal/mol RMSD 6.8 Ẳ αlow E = -783.53 kcal/mol RMSD 8.4 Ẳ β* E = -770.54 kcal/mol RMSD 0.9 Ẳ extlow2 E = -785.85 kcal/mol RMSD 7.3 Ẳ βlow2 E = -781.75 kcal/mol RMSD 6.4 Ẳ αlow2 E = -785.64 kcal/mol RMSD 8.4 Ẳ

17. 17 Protein G DIP results ext = -612.63 ± 9.31 kcal/mol 10.6 ± 0.6 Ẳ β = -638.42 ± 5.84 kcal/mol 1.6 ± 0.4 Ẳ α = -646.18 ± 6.90 kcal/mol 8.9 ± 0.2 Ẳ extlow = -640.60 ± 7.13 kcal/mol 9.1 ± 0.3 Ẳ β low = -651.05 ± 6.34 kcal/mol 6.9 ± 1.2 Ẳ α low = -646.14 ± 6.01 kcal/mol 9.0 ± 0.3 Ẳ extlow2 = -633.74 ± 7.13 kcal/mol 9.0 ± 0.7 Ẳ β low2 = -643.91 ± 7.86 kcal/mol 9.0 ± 0.3 Ẳ α low2 = -650.14 ± 6.33 kcal/mol 9.0 ± 0.2 Ẳ β* = -644.28 ± 6.08 kcal/mol 1.6 ± 0.2 Ẳ

18. 18 Protein G summary  β-hairpin stable at 300 K  Helical conformation lower in energy  Better energy compensation 3  Agreement with other simulation 4  Various factors may overstabilize helices (e.g., implicit solvent, salt bridges) 3 Muñoz, V.; Thompson, P. A.; Hofrichter, J.; Eaton, W. A. Nature, 1997, 390, 196-199. 4 Krivov, S. V.; Karplus, M. P. Natl. Acad. Sci., USA, 2004, 101, 14766-14770.

19. 19Conclusions  DIVE and DIP locate several PE minima  PES mapped by DIVE  PES of conformations at desired temperature with DIP  Conformations in good, if not excellent, agreement with experimental structures using DIP and DIVE

20. 20 Future directions  Continue validation of MD protocols with larger β-sheet  Further test MD protocols with tertiary structure  Predict structure of small protein

21. 21Acknowledgements  Ralph A. Wheeler  Zunnan Huang and Adam Hixson  National Research Service Award 5 F31 GM067560-03 to THC from the NIH/NIGMS  Oklahoma Center for the Advancement of Science and Technology (OCAST) HR01-148  Oklahoma Supercomputing Center for Education and Research (OSCER)  NSF/NRAC supercomputer time MCA96-N019