Date of Graduation

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Roger Koeppe

Committee Member

Neil Allison

Second Committee Member

Colin Heyes

Third Committee Member

Suresh Kumar

Fourth Committee Member

Frank Millett

Keywords

Pure sciences, Biological sciences, GWALP23, Histidine residues, Transmembrane peptides

Abstract

Designed transmembrane peptides were employed for investigations of histidine residues within the hydrophobic environment of the lipid bilayer by means of oriented solid-state deuterium NMR spectroscopy. Using the model peptide GWALP23 sequence (GGALW(LA)6LWLAGA) as a host framework, the effects of single and double histidine mutations were explored. Replacement of leucine residue 12 to polar neutral histidine had little influence on the peptide average orientation, however under strongly acidic pH conditions in DOPC bilayers, the histidine becomes positively charged (pKa 2.5) and the GWALP23-H12 peptide exits the membrane and adopts a surface-bound orientation. Conversely, mutation of leucine 14 to neutral histidine altered tilt direction and magnitude of the peptide by a similar extent that that previously observed for neutral lysine at position 14. In DOPC bilayers, when histidine 14 becomes positively charged (pKa 4.1), the peptide remains in a well-defined transmembrane orientation with an increased tilt value compared to the neutral form. In DLPC bilayers, no pH dependent behavior was observed for either GWALP23-H12 or GWALP23-H14. Mutation of alanine at position 13 to histidine caused no change in peptide orientation in DOPC bilayers under neutral pH conditions. When the pH is lowered to 2, the spectra contain multiple weak resonances, indicative of multi-state behavior for the charged GWALP23-H13 peptide. A leucine to histidine mutation at position 16, located directly below Trp19, causes a large change in the azimuthal rotation of the peptide of >100°. When the pH is lowered, histidine 16 becomes positively charged (pKa 3.5) and the tilt of the peptide increases. A peptide containing a pair of histidine residues at positions 12 and 13 displays behavior consistent with peptide oligmerization. The extent of this aggregation behavior appears to vary with pH, lipid composition, and macroscopic sample orientation. In DLPC bilayers, the presence of two histidine residues at positions 12 and 14 results in a decreased tilt value and a greater extent in helix unwinding. In DOPC bilayers, the GWALP23-H12,14 peptide orientation can not be explicitly identified. Additionally, unlike the single histidine peptides, the GWALP23-H12,14 does not display any pH-dependent behavior over a pH range of 2-7. Introducing a pair of histidine residues to positions 12 and 16 in DLPC bilayers results causes a change in helix tilt and rotation to allow both histidine residues access to the membrane interface. In DOPC bilayers, the GWALP23-H12,16 peptide adopts a surface-bound orientation which does not change with pH.

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