Date of Graduation

12-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Roger E. Koeppe II

Committee Member

Kathryn D. Curtin

Second Committee Member

Dan J. Davis

Third Committee Member

Francis S. Millett

Fourth Committee Member

William Oliver

Abstract

Model WALP peptides and "next generation" WALP-derived hydrophobic model peptides were employed to discover principles that govern protein-lipid interactions in biological membranes.

Ternary cholesterol-containing lipid mixtures were examined in the presence of WALP peptides of different lengths (acetyl-GWW(LA)nLWWA-ethanolamide, with n between 3 and 8). Deuterium NMR spectra from labeled lipids reveal that WALP peptides may stabilize lipid ordered "raft" domains and therefore promote lipid phase separation, albeit to a minor extent. The results depend upon whether dioleolyl- or diphytanoyl-phosphatidylcholine is present as the fluid lipid component.

Several WALP peptides were modified to remove anchoring Trp residues from one end or the other, thereby generating "half-anchored" WALP peptides which have aromatic anchor residues on only one side of the bilayer. The longer "half-anchored" WALP peptides (having 19-21 residues) were found to have small apparent average tilt values in DLPC, DMPC and DOPC lipid bilayer membranes. The results from a combined 15N PISEMA and 2H GALA analysis--with various analytical treatments of the peptide dynamics--confirmed the small average tilt angle for one of these peptides in DMPC bilayer membranes. Shorter "half-anchored" WALP peptides with a hydrophobic length theoretically capable of spanning only a monolayer leaflet, however, do not adopt well defined membrane orientations and often aggregate.

To a bilayer-spanning "half-anchored" WALP peptide having Trp17 and Trp18, we incorporated Trp or Arg as a "third" anchor in position 2 or 6. Incorporation of this third anchor increases the peptide tilt. When the third anchor is positioned at residue 6, the transmembrane conformation becomes destabilized.

In GWALP23, acetyl-GGALW(LA)6LWLAGA-ethanolamide, we incorporated Pro-12 (replacing Leu-12) within the transmembrane stretch of the peptide, introducing a distortion in the peptide alpha helix. Based upon combined 2H GALA and 15N PISEMA solid-state NMR experiments and analysis, the segments N-terminal and C-terminal to the proline are tilted by 34°-40° and 27°-29° (± 6°), respectively, with respect to the lipid bilayer normal, and the proline-induced kink angle is 20-23°.

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