The Latest from the NMR Collaboration Bicelles n’ Stuff
Group Meeting
October 23, 2008
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
How does alamethicin interact with the membrane?
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% D2O 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
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 NPzAT ensemble
(T = 323 K, Pz = 1 atm, A: fixed at end of eq.)
0 ns (after eq.)124 ns
NOE Stuff
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 CH2 groups of the lipid acyl chains as well as the terminal methyl group are separated
AA
NOESY spectrum of alm in bicelle
NOE Connectivities
(CH
2 ) 4-13(C
H2 ) 4-13
ring-H
20, H20
ba
ckb
on
e (N
H)3
-19
CH3
(NH2)7
(NH2)19
(NH)1
(CH3)3
(CH3)3
(CH
2 ) 4-13(C
H2 ) 4-13
ring-H
20, H20
ba
ckb
on
e (N
H)3
-19
CH3
(NH2)7
(NH2)19
(NH)1
(CH3)3
(CH3)3
MD Connectivities
ChoCH3
4 4 2 1 1 0 0 0 0 0 0 0 0 0 0 1 1 2 16 22 17
Acy
C2
7 2 3 2 2 1 2 0 1 2 0 0 1 0 1 4 2 0 30 24 16
AcyC3
7 2 2 2 2 1 2 0 1 1 0 0 1 0 1 2 1 0 25 27 14
Acy
C4-13
19 4 4 9 15 8 16 9 21 41 16 5 6 4 16 7 3 2 56 100 83
Acy
CH3
5 0 0 1 3 2 3 6 6 8 7 3 1 2 3 2 1 0 8 17 10
Aib1Aib3
Ala4 Ala6 Aib8 Aib10 Leu12 Val15 Aib17 Gln19 Gln7 Phl20Aib5 Gln7 Val9 Gly11 Aib13 Aib16 Glu18 Phl20 Gln19
0-1 1-3 3-6 6-10 10-15 15-20 20-30 30-50 50-100
backbone HN
NH2 HPhe
Should be redone with larger rlimit
strange
# proton contacts weighted by r -6
Helix Tilt (MD)
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0 10 20 30 40 50 60 70 80 90
helix tilt / deg.
PRE Stuff
PRE results
< 0.1 0.1-0.2 0.2-0.3 0.3-0.4 0.4-0.6 0.6-0.8 > 0.8 undet. 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.
Aib1Aib3
Gln7
Ala4
Aib5
Ala6
Val9
Aib8
Gly11
Aib10
Leu12
Val15Aib13
Aib16 Aib17
Phl20
Glu18
Gln19
Aib1Aib3
Gln7
Ala4
Aib5
Ala6
Val9
Aib8
Gly11
Aib10
Leu12
Val15Aib13
Aib16 Aib17
Phl20
Glu18
Gln19
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
75 40 45 44 15 8 4 0 1 4 1 0 0 0 2 4 3 3 79 100 47
Aib1Aib3
Ala4 Ala6 Aib8 Aib10 Leu12 Val15 Aib17 Gln19 Gln7 Phl20Aib5 Gln7 Val9 Gly11 Aib13 Aib16 Glu18 Phl20 Gln19
0-1 1-3 3-6 6-10 10-15 15-20 20-30 30-50 50-100
backbone HN
NH2 HPhe
Close to water interface
Far from water interface
But what about the cross peak to DHPC??
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
41
212
4
2
1
DPPC4Ctail
DLPC3Ctail
DHPC2Ctail
DMPC (C1-14) DHPC (C1-6)
Bicelles – how do they really look? 240 DHPC (2Ctail) 120 DMPC (3Ctail)
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 ??
A Volume Argument
bz
d / Å h / Å bz / Å Vbil/nm3 Vrim/nm3
140 40 0 616 310
140 40 10 531 395
140 30 10 398 233
90 30 10 150 154
100 50 0 393 374
bilayer part
rim part
DMPC (C1-14)DHPC (C1-6)
V(DHPC) > V(DMPC)/2
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
Simulations so far
360 2Ctail ; 12000 W
360 4Ctail ; 12000 W
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?