Date of Graduation

12-2019

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

Frank Millett

Second Committee Member

Colin D. Heyes

Third Committee Member

Susanne Striegler

Fourth Committee Member

Josh Sakon

Keywords

Helix unwinding, Histidine residue, Model membrane system, Protein-lipid interactions, Protein orientation and dynamics, Transmembrane peptides

Abstract

Since aromatic and charged residues are often present in various locations of transmembrane helices of integral membrane proteins, their impacts on the molecular properties of transmembrane proteins and their interactions with lipids are of particular interest in many studies. In this work, I used solid-state deuterium NMR spectroscopy in designed model peptide GWALP23 [GGALW(LA)6LWLAGA] with selective deuterium labels to addresses the pH dependence and influence of single and multiple “guest” histidine residues in the orientation and dynamic behaviors of transmembrane proteins. The mutations include Gly to His (G2/22 to H2/22), Trp to His (W5/19 to H5/19) and Leu to His (L8/16 to H8/16). For the glycine to histidine substitutions, either one or both, the peptides show similar biophysical properties to the host GWALP23 peptide, with modest motional averaging and tilted transmembrane orientations that scale with bilayer thicknesses. Yet, the dynamic motion about the average azimuthal rotation increases significantly when the helix carries only H22. However, when the tryptophan residues, W5 and/or W19 are replaced by histidines, the new histidine residues effectively anchor the transmembrane α-helix, providing similar transmembrane topology. A consistent ~30° shift in helix rotation is observed for Trp to His substitutions and found to be terminal-dependent. Modifying the core sequence of GWALP23 with His residues at positions 8 and 16 provides some interesting insights. The peptide is significantly tilted in DLPC, has multiple orientations in DMPC and surface bound in DPoPC and DOPC lipid bilayers, where the bilayer thicknesses increase consecutively from DLPC to DOPC. Further analysis for peptide with only H8 was performed. Results indicate multiple signal resonances, similar to -H8,16, but in a thicker lipid bilayer. Moreover, the helix with H8 alone significantly responds with pH in DLPC and DMPC lipids and two titration points for H8 was calculated. Finally, mutation of GWALP23 with two adjacent histidines at the N-terminal end (positions 4 and 5) causes a large increase in the motional averaging about helix azimuthal rotation, which in turn obscures the actual orientation and the peptide is found to adopt a very small tilt angle.

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