Studying Orientation and Conformation of a-helical Peptides at Interfaces.

Abstract

Membrane associated peptides and proteins with unique biological functions have drawn extensive attention due to their enormous therapeutic potential. Being an intrinsic surface-sensitive technique, Sum Frequency Generation (SFG) Spectroscopy has the capability to elucidate both structural and orientational information at biointerfaces, e.g., cell membranes. However, there are significant experimental and theoretical challenges in adapting this application from simple model peptides to more complex systems associated with cell membranes. My Ph.D. research started with elucidating structure and orientations of a simple linear helical peptide, Pep-1. By comparing its performance on gel-phase and liquid-phase lipid bilayers, we have elucidated the orientation information of Pep-1 at different concentrations and unveil the importance of lipid viscosity from a molecular level (Chapter 2). Unlike the linear peptide Pep-1, LL-37 is a bent α-helix and shows different structures on different kinds of lipids. We developed SFG orientation analysis methodology to address the bent issue. We then applied this methodology to more complex systems with mixed lipids and cholesterols to better approximate actual cells and discovered a “three-stage” interaction process in the presence of negative charged lipids (Chapter 3). We then studied the peptide GRK52-31, the active site of the GRK5 N-terminus together with its two truncated peptides. By combining SFG and ATR-FTIR, we found that the two parts of the peptide collaborate harmoniously in membrane interaction (Chapter 4). Previous work has been focused on analyzing the helical backbone and we further ask the question whether SFG is sensitive to detect the orientation of one single carbonyl group in the backbone to probe local structure of biomolecules at interfaces. We have successfully detected the SFG signal from a 13C=O labeled residue of ovisprin-1 on polymer surfaces. We simulated corresponding SFG spectra to match with the experiment result and concluded that the peptide is absorbed to the surface with the isotope-labeled residue buried in the hydrophobic interface (Chapter 5). Finally in Chapter 6, we demonstrated for the first time that, the isotope-labeled SFG technique we developed in Chapter 5 can serve as a new route for structure determination in situ on a single lipid bilayer.