Analysis of folding in pathways in Ac-ALA 5 -NH 2 peptide

1. Analysis of folding in pathways in Ac-ALA5-NH2 peptide Krzysztof Kuczera Departments of Chemistry and Molecular Biosciences, University of Kansas, Lawrence, KS 66045

2. Abstract . Molecular dynamics and replica-exchange simulations are performed for a model peptide, Ac-ALA5-NH2, using several popular force fields. The different models are used to calculate populations of helix, coil and intermediate states, melting curves, folding and nucleation times. The distributions of conformer populations are used to measure folding cooperativity. Interestingly, while the thermodynamic and kinetic predictions of the force fields differ widely, the calculated statistical properties of the helix-coil transition paths are qualitatively similar in all the studied models. References: [1] Garcia & Sanbonmatsu, Proc.Natl.Acad.Sci. USA99:2782 (2002) [2] Sorin & Pande, Biophys. J.88:2472 (2005) [3] Best et al., Biophys. J.95:L07 (2008) [4] Graf et al., J.Am.Chem.Soc. 129:1179 (2005)

3. Alanine-based peptide folding simulations • Replica exchange simulations by Garcia et al. showed exaggerated helix stability in AMBER99 [1] modified potential AMBER99GS • Simulation of  -helix folding kinetics by Pande also suggested the need for modified (,) potenial [2] • Hummer et al. showed that most popular force fields over-stabilze the –helix structure in short Ala-based peptides [3]

4. Experimental data on Alan • CD of Ac-Ala5-NH2 over 266-363 K  low helix content  5% at lowest temp. • NMR measurements of Alan , n=3-7  primarily PPII [4] [Gouri S. Jas, Baylor University, unpublished]

5. MD & REMD of Ac-Ala5-NH2 • OPLS/AA, G43A1, G53A6 and AMBER99SB give lowest helicity predictions • AMBER03, CHARMM22 - intermediate • AMBER99P, AMBERGS, CHARMM22/CMAP over-stabilize helix • REMD: melting not modeled well by most of the studied potentials REMD simulations: 32 replicas, 280-450 K, 30 Å cubic box with ca. 1000 waters, 100 ns NPT trajectory with GROMACS, for all except CHARMM potential MD: 1,000 ns NPT MD at 1 atm, 300 K with GROMACS CHARMM REMD: 40 ns in 37 Å bcc cell.

6. Folding of Ac-Ala5-NH2 : kinetics Predicted kinetic and equilibrium parameters span 2 orders of magnitude Sample OPLS/AA results

7. Folding of Ac-Ala5-NH2 : structures 001 111 000 100 011 010

8. Folding of Ac-Ala5-NH2 : patterns • Most FF : 000 dominant, very little 111, positive h-bond cooperativity • Populated intermediates: involve h-bonds #1 and #2 • Unusual: AMBERGS

9. Folding of Ac-Ala5-NH2 : pathways Rescaling time, smoothing and averaging reveals zipper-like transitions: - Unfolding initiated at C-terminus - Folding initiated at N-terminus

10. Conclusions • Predicted helix, coil and intermediate populations vary significantly between different models; most ff exaggerate helix population • Calculated folding, unfolding and nucleation rates span almost two orders of magnitude. Needed - experimental data to calibrate force fields. • Most ff predict that helical hydrogen bond formation is cooperative • Analysis of helix-coil transition paths indicates that all studied models predict a zipper-like mechanism, with unfolding initiated at C-terminus and folding initiated at N-terminus. • Unusual effects found: = persistence of the helical conformation at high temperatures in all models = increase of helix population with increasing T (AMBER99SB) = hydrogen bond anticooperativity (AMBERGS) Acknowledgments: The computer time and technical assistance from the Academic and Research Computing Services at Baylor University are gratefully acknowledged.

11. Folding of Ac-Ala5-NH2 : kinetics

12. Folding of Ac-Ala5-NH2 : kinetics