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NMR Techniques

New Solid State NMR Method

Mechanically Aligned Samples

Mechanically aligned samples are often used in conjunction with NMR to study peptides and lipids
in fluid lipid bilayers. However, certain peptides and lipids are difficult to align.
Phoshatidylethanolamines are prone to form inverted phases during hydration and the presence of
hydrophobic components makes fully hydrated bilayers difficult to make. We have recently reported
that the use of the sublimable solid naphthalene, during sample preparation, aids in the formation
of well-hydrated bilayers using these difficult components.
The details of this work can be found in Hallock, K.J., K. Henzler Wildman, D.K. Lee, and A.
Ramamoorthy. (2002). An innovative procedure using a sublimable solid to align lipid bilayers for
solid-state NMR studies. Biophys J 82:2499-503.

Lipids/peptide dissolved in
2:1 chloroform:methanol
with naphthalene.
Deposited and dried on glass
plates then taken for
vacuum drying.

Secret Ingredient
Naphthalene!

The initial sample has a lipid-peptide deposit that
also contains naphthalene.

Before Vacuum Drying

Glass plate

During vacuum drying, the naphthalene is removed because it has a vapor pressure of 50mTorr.
We think this results in a porous lipid-peptide deposit.

After Vacuum Drying

Glass plate

We think the porosity allows water to rapidly hydrate the lipid-peptide deposit, resulting in more
complete hydration. This method is particularly beneficial in preparing bilayers that contain
phosphatidylethanolamines, cholesterol and/or hydrophobic peptides like gramicidin.

Henzler Wildman, K.A. et al (reference below) have used E-coli Lipid Extract,
Avanti Product Number 100600 Component Percent/Wt. PE 67.0, PG 23.2, Cardiolipin 9.8
Some of the lipids suitable for this working method include:

DMPC Avanti Product Number 850345

POPC Avanti Product Number 850457

POPE Avanti Product Number 850757

POPG Avanti Product Number 840457

Avanti would like to thank Kevin J. Hallock for his kind assistance in the preparation of
this material. Dr. Hallock was Graduate Student Research Assistant, Chemistry
Department, at The University of Michigan.

Reference:
Henzler Wildman, K.A., D.K. Lee, and A. Ramamoorthy. (2003)
Mechanism of Lipid Bilayer Disruption by the Human Antimicrobial Peptide, LL-37.
Biochemistry 42:6545-58. [PubMed]
Abstract: LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site- specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.