1. ß-heptapeptide CC CC CC MD: rmsd 0.14 Å NMR structure (3 1 Helix) Reversibly folds on 10 -20 ns time scale (X. Daura et al.) 7 residues, in methanol (less atoms):  3000 at ~ 0.2 - 1 ns / hour (GROMACS)

2. ß-heptapeptide i) i)  -amino-acids (additional backbone carbon) ii) ii)Stable 2nd structure. iii) iii)Non-degradable peptide mimetics (e.g. highly selective somatastatin analogue) D. Seebach, B. Jaun + coworkers organic chem ETH-Zurich  -Heptapeptide (M) 31-helix in MeOH at 298 K (left-handed) Daura, X., Bernhard, J., Seebach, D., van Gunsteren, W. F. and Mark, A. E. (1998) J. Mol. Biol. 280, 925-932.

3. ß-heptapeptide unfoldfold unfold  -Heptapeptide, 340 K

4. ß-heptapeptide Starting structure  -Heptapeptide, 360 K

5. Protocol for MD: ß-heptatpetide 7 residues in 986 methanol molecules (~3000 atoms) GROMOS96 force field (van Gunsteren and co-workers) GROMACS software (www.gromacs.org) twin-range cutoff (1.0/1.4nm) for vdW and Elec. Reaction Field for long-range Elec. NP 1atm T and NV 300K T ensembles. Temp= 275.3, 286.4, 297.8, 322.1, 348.6, 399.6K Berendsen Thermostat, τ T =0.1ps and τ P =1.0ps run on 8 processors for 1ns/hour. ß-heptapeptide: Conventional MD

6. Time (ns) Folded = rmsd from NMR Model < 0.7 Å ß-heptapeptide: Conventional MD

7. Folded = rmsd from NMR Model < 0.7 Å ß-heptapeptide: Conventional MD

8. ß-heptapeptide: Replica Exchange Replica Exchange Replica Exchange

9. Protocol for the REMD: 20 structures selected as initial structures for the REMD 20 structures selected as initial structures for the REMD (more than 1ns spaced and rmsd from NMR > 0.3 nm) 200 ps of equilibration of each replica at its initial temperature 200 ps of equilibration of each replica at its initial temperature T= 275.3, 280.8, 286.8, 292.0, 297.8, 303.7, 309.7, 315.8, 322.1, 328.1, 335.0, T= 275.3, 280.8, 286.8, 292.0, 297.8, 303.7, 309.7, 315.8, 322.1, 328.1, 335.0, 341.6, 348.4, 355.4, 362.3, 369.5, 376.8, 384.2, 391.8, 399.6 341.6, 348.4, 355.4, 362.3, 369.5, 376.8, 384.2, 391.8, 399.6 V = Cste = V (NPT at 300K) V = Cste = V (NPT at 300K) exchange trials every 0.1, 0.5, 2.0 and 5.0 ps exchange trials every 0.1, 0.5, 2.0 and 5.0 ps 50 ns at 400 K to unfold it and generate random structures 50 ns at 400 K to unfold it and generate random structures ß-heptatpetide 7 residues in 986 methanol molecules (3000 atoms) ß-heptatpetide 7 residues in 986 methanol molecules (3000 atoms) T controled by a Berendsen bath, τ T =0.1ps. T controled by a Berendsen bath, τ T =0.1ps. ß-heptapeptide: Replica Exchange

10. RMSD from the NMR model (C  res. 2-6) Exchange trial every 2.0 ps ß-heptapeptide: Replica Exchange

11. RMSD from the NMR model (C  res. 2-6) 0.5ps2.0ps5.0ps Time of Equilibration Increasing with Time Interval Between Exchange Trial Ratio of Folded Peptide at each temperature Criterion: folded = rmsd  0.7Å ß-heptapeptide: Replica Exchange

12. Folded Peptide ratios as a function of the interval between exchanges ß-heptapeptide: Replica Exchange

13. The replicas explore temperatures at different rates. Temperature Exploration 0.5ps 2.0ps 5.0ps ß-heptapeptide: Replica Exchange

14. Simulations converged Folded Peptide ratios as function of the interval between exchanges ß-heptapeptide: Replica Exchange

15. Folded Peptide ratios: REMD vs. Standard MD ß-heptapeptide: Replica Exchange

16. Folding free energy: REMD vs. Brute Force MD ß-heptapeptide: Replica Exchange

17. 72 9 3 2 40 13 9 255 9 4 428 14 7 323 8 5 3 1 2 3 4 1 2 3 4 50 15 9 3 60 10 3 2 70 6 3 231 12 7 326 10 8 5 8 7 4 448 17 8 3 69 13 7 2 61 11 8 243 10 7 329 8 7 413 5 5 565 12 7 2 NPT NVT REMD 275286298 322348 399 Cluster # T (K) Conformational exploration: REMD vs. Brute Force MD 72 9 3 2 40 13 9 255 9 4 428 14 7 323 8 5 350 15 9 3 60 10 3 2 70 6 3 231 12 7 326 10 8 5 8 7 4 448 17 8 3 69 13 7 2 61 11 8 243 10 7 329 8 7 413 5 5 565 12 7 2 Cluster Analysis: use of the 4 more populated clusters (% of the total ensemble) REMD simulations reproduce conformational ensembles ß-heptapeptide: Replica Exchange

18. REMD simulation: 1) accurately reproduces - thermodynamics (folding free energy) - conformational (clusters) MD data for folding 2) faster than MD simulation timeREMD: 20*15ns = 300ns BFMD: 2*200+400+3*800 =3200ns one TempREMD: 20*15ns = 300ns BFMD: 1*800ns = 800ns real timeREMD: 15ns / 1 CPU = 7.5 days BFMD: 800ns / 8 CPUs = 34 days Efficiency: REMD vs. Brute Force MD ß-heptapeptide: Replica Exchange