Proteins Inside Cells and On the Surface of Membranes: Developing Three Dimensional In-Cell NMR and Structural Characterization of a Trimeric Membrane Proximal External Region Construct from HIV-1 gp41

Abstract

Our ability to understand complex biological processes is enhanced by studying the components of those processes at atomic resolution. Major advancements in technology and methodology have been made that continue to improve our ability to determine atomic resolution structures of proteins. These advancements have also enabled new ways to study difficult protein systems. In this dissertation, I will discuss our efforts to apply NMR spectroscopy and other biophysical techniques to better understand two problems. The first problem, and the focus of the first part of this work, is to understand the behavior of proteins in the complex milieu of a living cell. We have developed 3D In-Cell protein NMR and demonstrated its use to assign the backbone resonances of a protein in living E. coli cells. I will also discuss our application of In-Cell NMR to show that the methionine repressor, MetJ, is generally associated non-specifically with DNA inside living cells. In the second part of this dissertation I will discuss our efforts to better understand the biophysical behavior of the membrane proximal external region (MPER) of the HIV-1 envelope protein, gp41. This region is membrane associated and an important target for HIV-1 vaccine development because it contains the epitopes for several of the broadly neutralizing antibodies against HIV-1. In addition to studies of the monomeric peptide, we have developed a new trimeric MPER construct, designated gp41-M-MAT, that associates with detergent micelles and lipid bilayers. This construct is a stable trimer, which binds the broadly neutralizing antibodies 2F5 and 4E10, as shown by equilibrium analytical ultracentrifugation and surface plasmon resonance. Finally, we have solved the structure of this construct bound to detergent micelles using NMR spectroscopy. The structure shows that the MPER adopts an α-helix conformation and suggests that each helix in the symmetric trimer associates directly with the surface of detergents and lipid membranes. This association suggests that the MPER might contribute to the mechanism of viral fusion by inducing strain in the viral membrane.