Date of Graduation
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Roger E. Koeppe II
Suresh Kumar Thallapuranam
Second Committee Member
Ralph L. Henry
Third Committee Member
David S. Mcnabb
Fourth Committee Member
Pure sciences; Biological sciences; GWALP23; Glutamic acid; Membrane biophysics; Transmembrane helix
The molecular properties of transmembrane proteins and their interactions with lipids regulate biological function. Of particular interest are interfacial aromatic residues and charged residues in the core helix whose functions range from stabilizing the native structure to regulating ion channels. This dissertation addresses the pH dependence and influence of potentially negatively charged tyrosine, glutamic acid or aspartic acid side chains. We have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as favorable host peptide framework. We have substituted W5 with Tyr (Y5GWALP23) and Leu residues with Glu (L12E, L14E or L16E) or Asp (L14D or L16D), and have incorporated specific 2H-labeled alanine residues within the core helix or near the ends of the sequence. Solid-state 2H-NMR spectra reveal a pKa of about 10.5 for bilayer incorporated Y5GWALP23.
Solid-state 2H-NMR spectra of GWALP23-E12, –E14 and –E16 with core labels reveal little change to the orientation of the transmembrane helix over a pH range of 4 to 12.5 but modest changes in quadrupolar splitting magnitudes above pH 12.5 in DLPC bilayer membranes, with E12 peptides showing no change even at pH 13. The E12, E14 and E16 peptides display broad 2H NMR spectra in aligned DOPC bilayers, with individual resonances not being observed for the core labels. Labeling the ends of the helix at A3 and A21 provided insights into the pH-dependent unwinding of the E14 and E16 peptide helices in both lipid systems.
An aspartic acid residue at position 14 shows contrasting behavior to that of its Glu counterpart. The 2H-NMR spectra for core 2H-alanines of GWALP23-D14 show a preference for a well oriented conformation in DOPC bilayers in comparison to DLPC lipids. While the core helix does not respond to pH, the helix terminals show changes in unwinding between pH 6 and 13 suggesting a possible pKa around 13. The polar but uncharged Gln residue at position 14 behaves similarly to Glu in DLPC and DOPC lipid bilayers. The Q14 peptide, however, does not titrate in either lipid and displays well-resolved sharper 2H-NMR resonances in DLPC bilayers. The combined results illustrate complex behavior for carboxyl and carboxamide side chains in bilayer membranes.
Rajagopalan, Venkatesan, "Influence of pH and Acidic Side Chain Charges on the Behavior of Designed Model Peptides in Lipid Bilayer Membranes" (2016). Theses and Dissertations. 1791.