1. The Latest from the NMR Collaboration Bicelles n’ Stuff Group Meeting October 23, 2008

2. 20 amino acid antimicrobiel peptide. Part of the immune system (for fungi). Forms channels in membranes which allow water and ions to go through. Destroys membrane potential. Alamethicin Motivation: Potentially a good and simple membrane channel model Insight into this family of proteins could be valuable in the development of antibiotics Gln7 Glu18 Gln19

3. How does alamethicin interact with the membrane?

4. Liquid NMR Setup 1 M stock solutions of DMPC and DHPC in 10 mM phosphate buffer at pH 6.6. Mixed with lyophilized alamethicin, repeated cycles of vortexing and centrifugation. DMPC and phosphate buffer added to yield a solution with 300 mM total lipid concentration and 15 mM peptide. 10% D 2 O added for field lock. Vortexed and centrifuged several times until clear solution. NOE : lipid-peptide hydrogen interactions PRE : Paramagnetic Relaxation Enhancement. Gadodiamide (gadolinium complex) remains in solution. Affects the relaxation in a distance dependent manner => tells where the different hydrogens are positioned in the bilayer. Sample Preparation Output

5. AA MD of Alamethicin in DMPC 25 peptides and 330 lipids + water ~125,000 atoms. Energy-minimized and equilibrated for 200 ps with peptides fixed. Energy-minimized and equilibrated for 1 ns in the NPT ensemble (T = 323 K, P = 1 atm, NAMD and CHARMM FF) Production run of 100+ ns in the NP z AT ensemble (T = 323 K, P z = 1 atm, A: fixed at end of eq.) 0 ns (after eq.) 124 ns

6. NOE Stuff

7. Difficult to Interpret Spectra Absence of Hα protons and NMR-resolvable side-chain protons in Aib Two prolines => lack of amide protons Lipid acyl groups (positions 4-13) are degenerated to one resonance at 1.28 ppm => Only first and second CH 2 groups of the lipid acyl chains as well as the terminal methyl group are separated NOESY spectrum of alm in bicelle

8. NOE Connectivities

9. MD Connectivities Cho CH3 442110000000000112162217 Acy C2 723221201200101420302416 Acy C3 722221201100101210252714 Acy C4-13 19449158169214116564 7325610083 Acy CH3 50013236687312321081710 Aib1 Aib3 Ala4Ala6Aib8Aib10Leu12Val15Aib17Gln19Gln7Phl20 Aib5Gln7Val9Gly11Aib13Aib16Glu18Phl20Gln19 0-11-33-66-1010-1515-2020-3030-5050-100 backbone H N NH 2 H Phe Should be redone with larger r limit strange # proton contacts weighted by r -6

10. Helix Tilt (MD)

11. PRE Stuff

12. PRE results < 0.10.1-0.20.2-0.30.3-0.40.4-0.60.6-0.8> 0.8undet.non-protons Close to water interface Far from water interface s -1 Is this alamethicin structure found from the same type of experiment? An average structure could be constructed from the MD sim as well.

13. MD Peptide-Water Contacts If I made this data for the lipids as well, I bet it would not look as nice as the NMR results # peptide proton-water oxygen contacts weighted by r --6 754045441584014100024337910047 Aib1 Aib3 Ala4Ala6Aib8Aib10Leu12Val15Aib17Gln19Gln7Phl20 Aib5Gln7Val9Gly11Aib13Aib16Glu18Phl20Gln19 0-11-33-66-1010-1515-2020-3030-5050-100 backbone H N NH 2 H Phe Close to water interface Far from water interface

14. But what about the cross peak to DHPC??

15. Main Classes : Nonpolar (N), Charged (Q), Apolar (C), Polar (P) Subclasses (N,Q) : Hydrogen bond donor (d), acceptor (a), both (da), none (0) Subclasses (C,P) : Degree of polarity (1-5) MARTINI CG Class C – apolar NAMD: Class Nda – nonpolar and hydrogen bond donor and acceptor MARTINI: Class P – polar Class Na – nonpolar and hydrogen bond acceptor Class Qa – charged and hydrogen bond acceptor Class Q0 – charged 4 4 1 2 1 2 4 2 1 DPPC 4Ctail DLPC 3Ctail DHPC 2Ctail DMPC (C1-14)DHPC (C1-6)

16. Bicelles – how do they really look? 240 DHPC (2Ctail) 120 DMPC (3Ctail)

17. Simulations so far q = [DMPC]/[DHPC] = long/short = 0.5 120 3Ctail ; 240 2Ctail ; 12000 W 6 Alm ; 120 3Ctail ; 240 2Ctail ; 12000 W 120 3Ctail ; 240 2Ctail ; 48000 W 120 4Ctail ; 240 2Ctail ; 12000 W Previous slide 1Ctail ??

18. A Volume Argument bz d / Åh / Åbz / ÅV bil /nm 3 V rim /nm 3 140400616310 1404010531395 1403010398233 903010150154 100500393374 bil ayer part rim part DMPC (C1-14)DHPC (C1-6) V(DHPC) > V(DMPC)/2

19. Simulations so far q = [DMPC]/[DHPC] = long/short = 3.2 768 3Ctail ; 240 2Ctail ; 31500 W 768 4Ctail ; 240 2Ctail ; 31500 W 384 4Ctail ; 120 2Ctail ; 16000 W ~half size

20. Simulations so far 360 2Ctail ; 12000 W 360 4Ctail ; 12000 W

21. Random Thoughts so far Short and long lipids mix!! I would like to see cases where they actually don’t q = 0.5 => bicelle/micelles q = 3.2 => ~bilayers What about other q’s ? Can we find the limits where bicelle go to bilayer and so on. The more lipid and/or water included in the simulation, the slower convergence to low energy structure => can we just say it is fair to look at small systems? Equal distribution of the short lipids to relieve potential stress? Does the distribution of the lipids change over time? Relevant simulations should be repeated many times. Is the CG model of DHPC a good description – check the literature Is there a problem in representing DHPC with only one bead in each lipid tail?? Artifacts from number of lipids involved in simulation should be investigated. Dependence of the amount of water should be investigated. Reverse CG or simply redo in AA for the most interesting set-ups to validate CG Could something beside lipids in the NMR mixture affect size/shape/segregation? Could the NMR signals be explained by something else than the perfect-model bicelle based on the simulations? What type of experiments have been done on bicelles, and what can we really be sure of? In lab it takes several hours for the lipid mixture to obtain the ”right” macro structures => several close minima that actually have different physical properties?