Date of Graduation

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Roger E. Koeppe II

Committee Member

James F. Hinton

Second Committee Member

Francis S. Millett

Keywords

Anchoring amino acids, Arginine, Deuterium NMR, Protein-lipid interactions, Solid-state NMR, Transmembrane peptides

Abstract

Designed transmembrane peptides were employed for investigations of protein-lipid interactions by means of oriented solid-state deuterium NMR spectroscopy using isotope-enriched alanine residues. Using the model GWALP23 sequence (GGALW(LA)6LWLAGA) as a host peptide having single interfacial tryptophan anchor residues, the effects of different guest mutations were explored. Replacements of glycine residues 2 and 22 to positively charged lysine or arginine on both termini had little influence on the peptide average orientation. Conversely, glycine to tryptophan substitutions had profound effects, manifested in the increased dynamics and altered tilt direction of the peptide. While the charged residues at the peptide termini did not cause significant changes relative to the GWALP23 sequence, leucine to arginine mutations close to the peptide center led to dramatic consequences. Thus GWALP23-R14 retained a transmembrane topology, with the orientation and dynamics largely governed by the arginine residue, while GWALP23-R12 adopted multistate behavior in DOPC, with both transmembrane and interfacial states being populated. Coarse-grained molecular dynamics simulations, performed by collaborators, yielded substantial agreement concerning the interactions among arginine, tryptophan and lipid bilayers. Further insights into the multistate behavior of GWALP23-R12 were acquired by altering the host sequence to the isomeric GW3,21ALP23, which offers a longer separation between the tryptophan anchor residues. Both the L12R and L14R mutants of this modified sequence retained transmembrane topology, suggesting that the unique arrangement of tryptophan and arginine residues in GWALP23-R12 is responsible for its multistate character. In addition to serving as a host sequence, GWALP23 itself was modified for an investigation of hydrophobic matching, by shifting the tryptophan residues outward toward the termini (GW3,21ALP23) or inward toward the center (GW7,17ALP23), leading to peptide isomers with identical amino acid composition, but different effective hydrophobic (inter-Trp) lengths. In addition to altered tilt angles, tryptophan side chain reorientation was investigated and was found to provide additional response to hydrophobic mismatch conditions. In selected cases the 2H NMR data were analyzed in conjunction with restraints from separated local-field 15N solid-state NMR spectra. The combined analysis of the 2H and 15N NMR data provided multiple constraints and proved advantageous for explicit modeling of the peptide dynamics.

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