Computer simulations of polypeptides in carbon nanotube

1. Computer simulations of polypeptides in carbon nanotube Seneviratne Samaratunga Ph.D. Thesis Defense Advisor: Professor Jay C. Rasaiah Department of Chemistry July 2, 2013

2. Outline • Motivation and objectives • Introduction • Questions to be addressed • Previous related work • My project in confined spaces • Method of study • Results and summary • Conclusions and future work • Acknowledgements 1

3. Motivation and objectives • Motivation • The mechanism of polypeptide formation of secondary structures within the ribosomal exit tunnel remains one of the most important unsolved problems in biophysical chemistry • Objectives • The main objective of this research is to characterize protein behavior inside the ribosome tunnel to assist nanomedicine in terms of drug design and delivery, and ultimately support for natural human biological structure and function 2

4. Helix formation within the ribosomal exit tunnel S. Bhushan, M. Gartmann and M. Halic, Nature Structural & Molecular Biology2010, 17 , pp313-317

5. Protein structure Carboxyl terminus Amino terminus Torsion angle, (C – Cα) and Ø(Cα – N) 3 Cox, Lehninger Principles in Biochemistry, chapter 6

6. The alpha-helix • 3.613 helix Amino terminus Cα8 Cα9 Cα7 Cα5 3.613 helix Cα6 R-groups spiraling out from central axis The pitch of a standard helix is 5.4 Å Cα4 Cα3 Cα1 Cα2 Carboxyl terminus a-helix have conformations with j = –45–50°, and f = –60° Helix dipole structure 1. Cox, Lehninger Principles in Biochemistry 4 2. http://www.docstoc.com/docs/18305133/protein-conformation

7. What is ribosome and its exit tunnel? Exit Tunnel 50S Subunit Proteins 50S Subunit Exit tunnel 30S Subunit Proteins 30S Subunit mRNA Exit tunnel Constriction site Active site (PTC) mRNA D.V.Fedyukina and S.Cavagnero , Annu. Rev. Biophys. 2011. 40, pp337–359 5

8. Questions to be addressed concerning the polypeptide exit tunnel as confined system • What are the forces that drive helix formation within the tunnel? • Is there a critical diameter or is there a range of diameters within which the alpha helices are formed? • How do the interactions of peptides along the tunnel walls affect helix formation? • How long does the peptide remain in the tunnel before it makes a tertiary structure at the end of the tunnel? 6

9. Previous work • Sorin and Pande (MD) 23-residue polypeptide • O’Brien simulated polypeptides confined to various sizes of carbon nanotubes • Zhou presented a theory in 2007 to describe the activity of confined water • Thirumalai and Vaitheeswaran determined factors affecting peptide conformation 7

10. Work done by Sorin and Pande D eff = D - 2S2 E. J. Sorin and V. S. Pande, J. Am. Chem. Soc. 2006, 128, pp6316-6317 8

11. Poly- Alanine,  = 0.01 Poly- Alanine,  = 1.0 Helix stability depends on nanotube peptide interaction Entropic stabilization Enthalpic destabilization E. P. O’Brien, G. Stan, D. Thirumalai, and B. R. Brooks, Nano Lett., 2008, 8 , pp3702-3708 9

12. My research Molecular dynamics study of -helix formation in a CNT with 23-residue polyalanine and [Glu-(Ala)3-Lys]5 polypeptide to mimic the effect of confinement in the ribosome tunnel.

13. Method : Molecular Dynamics • A computational method describing equilibrium and dynamic properties of a biological system • Generates system configurations by integrating Newton’s laws of motion to calculate the time dependence parameters • Simulations can be carried out that are difficult or impossible in the laboratory • Can be used to explore the macroscopic properties of a system through microscopic simulations • Connects structure and function by providing additional information to X-ray crystallography and NMR 10

14. Newton’s Second Law of Motion and integration algorithms r(t + δt) = 2r(t) – r(t –δt) + a(t) δt2 Verlet algorithm The Leap-frog algorithm r(t + δt) = r(t) + v(t +1/2δt)δt 11

15. Force field used Θ Φ r General form of all-atom force fields The most time- consuming part. H-bonding term Van der Waals term Electrostatic term A.D. MacKerell, J. Comp. Chem. 2004 ,25, pp1584-1604 12

16. System preparation • Prepare PDB and PSF files of CNT • Prepare PDB and PSF files of polypeptide • Combine polypeptide and CNT • Adjust the peptide carefully inside the nanotube • Add water box with suitable dimensions • Combine system with water box • Apply hexagonal boundary conditions for the system • Fix six atoms around the carbon nanotube • Simulate the system 13

17. Part I Some important system structures Alaninehomopolymer after solvating with 29181 atoms of TIP3P water An image showing the hexagonal shape of the boundary Two images shown with the fixed atoms displayed with their Van der Walls radius. 14

18. Simulation trajectories obtained S.Samaratunga, D.Suvlu ,D.Thirumalai and J.C.Rasaiah 2013 in prep 15

19. Snapshots of two different values of   = 0.56  = 0.64 D = 13.6Å D = 14.9Å D = 16.3Å Left handed - helices 16

20. Mean helical content vs. tube diameter from simulation for capped homo polyalanine S.Samaratunga, D.Suvlu ,D.Thirumalai and J.C.Rasaiah 2013 in prep 17

21. Number of water molecules within 3.5Å from the peptide S.Samaratunga, D.Suvlu ,D.Thirumalai and J.C.Rasaiah 2013 in prep 18

22. Mean helical content vs. time 0.8 Mean Helical Content Time (ns) Time (ns) When the value of  was less than unity there was a sudden reversal in probability of forming -helix with two different diameter nanotubes. 19

23. Dipole autocorrelation functions when = 1.0 20

24. Probability of center of mass of peptides and CNT 21

25. Comparison of phase diagrams A B 1: D = 14.9 and = 1.0, 2: D = 13.6 and = 0.56 3: D = 10.9 and = 1.0, 0.64 and 0.56 4: D = 13.6 and = 0.58 5: D = 25.8 and = 1.0, 0.8, 0.64 and 0.56 A: O’Brien work B: My work S.Samaratunga, D.Suvlu ,D.Thirumalai and J.C.Rasaiah 2013 in prep 22

26. Part II Snapshots of two different  for peptide in chiral CNT  = 1.0  = 0.56 D = 14.3Å 11, 10 D = 15.0Å 12, 10 D = 12.9Å 9, 10 D = 17.1Å 15, 10 S.Samaratunga, D.Suvlu ,D.Thirumalai and J.C.Rasaiah 2013 in prep 23

27. 23-residue polypeptide in chiral CNT S.Samaratunga, D.Suvlu, D.Thirumalai and J.C.Rasaiah 2013 in prep 24

28. Mean helical content vs. time When the value of  was less than unity there was a reversal in probability of forming -helix with two different diameter nanotubes. 25

29. Ramachandran plot of peptide in chiral CNT 26

30. Phase diagram in the (λ, D) plane 27

31. Part III 25-residue [Glu-(Ala)3-Lys]5 peptide in CNT D = 14.9Å D = 25.8Å D = 16.3Å D = 25.8Å D = 40.9Å 28

32. Plots of 25-residue [Glu-(Ala)3-Lys]5 peptide in CNT • Ramachandran plot shows when D= 25.8Å there is no helix formation • The number of water molecules increases as the diameter increases 29

33. Alaninehomopolymer(23 capped) Animation of helix formation of alaninehomopolymer Diameter of CNT= 14.9Å 30

34. Summary • Alpha helices are formed in the ribosome tunnel which is a confined system • Alaninehomopolymer (A-23-capped) forms an -helix in a 14.9Å diameter CNT when = 1.0 • My results are different from Pande and Sorin’s • When the  value change is less than one: • Highest helicity shifted to D = 13.6 Å • I observed left handed -helix when D = 16.3 Å • A-23-capped polyalanine forms -helix in critical diameter between 12.9 and 14.3Å in chiral CNT 31

35. Conclusions • The tunnel has a non-uniform diameter. We can assume that there should be a secondary structure formed throughout the lower part of the tunnel regardless of the diameter at each point. • At each point the strength of the interaction should vary to maintain helical structure. Electronic properties are not the same along the tunnel. • If the peptide cannot form its secondary structure, the result would be straight-chain polymer released at the end of the exit tunnel, having no possibility to form a tertiary structure, and thereby causing disease. 32

36. Future work • Use more accurate and detailed simulations with Replica Exchange Molecular Dynamics (REMD) • Introduce experimental parameters, such as temperature or pH, to be incorporated and used in algorithms • Conduct molecular dynamics simulation on potassium channels which conduct K+ ions across the cell membrane • Prepare biosensors by modifying surface of the Carbon Nanotube with Ionic Liquids and polymers 33

37. References [1] G.Ziv, G. Haran and D. Thirumalai,Proc. Natl. Acad. Sci., 2005, 102, pp18956-18961. [2] M.S.Cheung and D. Thirumalai,J. Mol. Biol.,2006, 357, pp632-643. [3] E.J.Sorin and V.S. Pande,J. Am. Chem. Soc., 2006, 128, pp6316-6317. [4] D.Lucent, V. Vishal and V.S. Pande,Proc. Natl. Acad. Sci.,2007, 104, pp10430-10434. [5] E.P.O’Brien, D. Thirumalai and B.R. Brooks,NanoLett., 2008, 8, pp3702-3708. [6] S.Vaitheeswaran and D. Thirumalai,Proc. Natl. Acad. Sci.,2008, 105, pp17636-17641. [7] R.I.Dima and D. Thirumalai, Proc. Natl. Acad. Sci.,2004, 101, pp15335-15340. [8] D.V.Fedyukina and S.Cavagnero , Annu. Rev. Biophys, 2011, 40, pp337–359 [9] M. R.Betancourt and D. Thirumalai,J. Mol. Biol.,1999, 287, pp627–644. [10] H. X.Zhou and K.A. Dill,Biochemistry,2001, 40, pp11289–11293. [11] D.K.Klimov,D. Newfield and D. Thirumalai,Proc. Natl. Acad. Sci.,2002, 99, pp8019–8024. [12]A. P. Minton, J.Biophys, 1992, 63, pp1090–1100. [13] G.Hummer, J.C. Rasaiah and J.P. Noworyta,Nature,2001, 414, pp188-190. [14] J.C.Rasaiah, S. Garde and G. Hummer,Ann. Rev. Phys. Chem.,2008, 59, pp713-740 [15] C. Deeker , Nature Nanotechnology,2007,2, pp209-215. [16] S.Samaratunga, D.Thirumalai and J.C.Rasaiah 2013 in prep

38. Acknowledgments • Advisory Committee • Dr. Jayendran C. Rasaiah • Dr. Howard H. Patterson • Dr. Scott Collins • Dr. Barbara J. W. Cole • Dr. Yifeng Zhu • Group members • Family and friends

39. Thank you

40. The PDB file- text format

41. Peptide synthesis

42. Topics for discussion • Motivation and objectives • Introduction • Carbon nanotube • Structure of the peptide bond • Ribosome and its exit tunnel • Previous work • My project in confined spaces • Summary and future work 1

43. Difference between Charmm and Amber force fields Amber parameters are not physically sound. The charges are based on the restrained electrostatic potential derived from gas-phase quantum mechanical calculations at the HF/6-31G* level. These charges are very sensitive to conformational changes, so the amber parameters are not reliable. For example, the backbone charges are different for each amino-acid, yet there's only one set of atom types for the N, C, Ca and O atoms (with associated bond, angle and dihedral terms). In particular the dihedral angle parameters may yield peculiar results. CHARMM has a more robust parameter set.

44. Ψ(psi) and Ø(phi) angle ranges for helicity calculation Based on the properties of a standard helix PHI_MAX = -57.0 + 30.0 PHI_MIN = -57.0 - 30.0 PSI_MAX = -47.0 + 30.0 PSI_MIN = -47.0 - 30.0 Based on the Klimovdefinition PHI_MAX = -48.0 + 30.0 PHI_MIN = -80.0 - 30.0 PSI_MAX = -27.0 + 30.0 PSI_MIN = -59.0 - 30.0 • How to stabilize the alpha helix: • Intrachain H-bonding • Minimization of steric interference D.K. Klimov and D. ThirumalaiStructure, 2003, 11, pp295–307