Date of Graduation

12-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor

Roger Koeppe, II

Committee Member

Stefan Kilyanek

Second Committee Member

Wesley Stites

Third Committee Member

Susanne Striegler

Fourth Committee Member

Suresh Thallapuranam

Keywords

2H NMR, GWALP23, Membrane Biophysics, Solid-State NMR

Abstract

Designed model transmembrane peptides and oriented 2H and 15N solid-state nuclear magnetic resonance (NMR) spectroscopy were used to analyze how simple sequence modifications can influence peptide structure, behavior and dynamics as well as for determining the pKa of glutamic acid at the membrane interface. The GW5,19ALP23 (acetyl-GGALW(LA)6LWLAGA-amide) peptide framework adopts a well-defined tilted orientation in lipid bilayers (DLPC, DMPC and DOPC) and undergoes low amounts of dynamic motion. The sequence was initially modified by moving the Trp residues outwards to positions 4 and 20. This new sequence GW4,20ALP23 (acetyl-GGAW(AL)7AWAGA-amide) displays high amounts of signal averaging of NMR observables caused by extensive dynamic motion about its average azimuthal rotation. The high dynamics are due to side chain competition induced by the opposing radial locations of the interfacial Trp(W) residues. The GW4,20ALP23 sequence was subsequently modified by introducing Arg(R) residues at either position 14 or 12. The R14 peptide adopts a well-defined tilt in lipid bilayers while completely arresting the high dynamics of the parent framework. In response, the C-terminal Trp causes partial unwinding of the core helix, while the N-terminal residues tighten into the core helix to compensate. R12 pulls the peptide to the membrane surface. A helix discontinuity is observed beginning at residue 11 as well as the formation of a partial N-terminal 310-helix. Modifying the core sequence of GW4,20ALP23 with Leu residues at positions 5 and 19 does not significantly affect the high dynamics, yet causes the peptide to adopt the same tilt as the original GW5,19ALP23 sequence. Removing W4 and replacing it with two Phe residues at positions 4 and 5 not only reduces the dynamics but also causes C-terminal helix distortion. Moving away from helix dynamics, 2H NMR was used to determine the side chain pKa of an interfacial Glu residue in the GW5,19ALP23 framework will oriented in the three lipid bilayers. The pKa increases with lipid bilayer thickness ranging from 4.3 to 11.0. Together, these experiments with model membrane peptides and solid-state NMR can be used to help our understanding of the basic principles that govern protein-lipid interactions.

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