Solid-state NMR studies of structure and dynamics of HIV-1 capsid (CA) protein assemblies

Date
2013
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
There are around 34 million people in the world living with HIV-1, which is a causative agent of acquired immunodeficiency syndrome (AIDS). AIDS has become the ninth leading cause for death of people ages 25-34 in the US. Even though highly active antiretroviral therapy (HAART) showed effectiveness in suppressing the virus replication and significantly prolonged the patients lives, AIDS still remains an uncured disease. To develop new therapies, atomic-level understanding of the mechanism of HIV-1 lifecycle, including the structures of the various protein assemblies, is needed. The Gag polyprotein and its component capsid (CA) protein are essential constituents of the HIV-1 life cycle, and have recently attracted attention as targets for drug development. However, the atomic resolution structure and the dynamics of Gag and CA protein assemblies and their complexes with small-molecule inhibitors are not available because these assemblies are not amenable for characterization by traditional structural biology methods, X-ray diffraction and solution NMR spectroscopy. Solid-state NMR spectroscopy has the unique capability of providing atomic-level structural and dynamics information in large protein assemblies. The focus of this dissertation is establishing solid-state NMR spectroscopy as an atomic-level probe of structure and dynamics in HIV-1 protein assemblies. This effort required first establishing sample conditions for formation of HIV-1 protein assemblies that give rise to high-resolution solid-state NMR spectra for subsequent structural studies. In my Ph.D. work, I have optimized protocols to prepare homogeneous HIV-1 CA protein assemblies in vitro, developed confocal imaging method to characterize the morphologies of the resulting assemblies. With the suitable samples in hand, I have acquired solid-state NMR spectra on assemblies of CA protein and the maturation intermediate, CA-SP1, for structural analysis. Using these solid-state NMR data, I and my colleagues have obtained novel insights into the following aspects of HIV-1 structural biology: i) conformation of CA protein in conical assemblies; ii) the role of conformational dynamics of the hinge region in the structural polymorphism of CA in conical assemblies; iii) conformation of spacer peptide SP1 in tubular CA-SP1 assemblies; iv) conformation and dynamics of CA in tubular assemblies. In this thesis, I will first discuss the preparation and characterization of HIV-1 CA and CA-SP1 protein assemblies (Chapter 2 and 3). I will then describe the resonance assignments and secondary structure analysis of conical assemblies of HIV-1 CA protein (Chapter 4). Next, in Chapter 5, I will present the dynamics studies of conical assemblies of HIV-1 CA protein assemblies. In Chapter 6, I will discuss the resonance assignments and conformational analysis of tubular assemblies of HIV-1 CA and CA-SP1 proteins. The long-term goal of this research is developing comprehensive understanding of the mechanism of capsid assembly and disassembly, HIV-1 maturation, through the structural and dynamics analysis of the CA and Gag assemblies. The work discussed here represents the first step toward this goal and lays out the methodological and intellectual foundations enabling the solid-state NMR analysis of HIV-1 protein assemblies.
Description
Keywords
Citation