Insights into the structure and dynamics of HIV-1 maturation intermediates and nonuniform sampling methods for magic angle spinning NMR

Polenova, TatyanaA key step in the HIV-1 lifecycle is viral maturation, which proceeds through the proteolytic cleavage of the Gag Polyprotein into its constituent domains, including: matrix (MA), capsid (CA), nucleocapsid (NC), P6, and two small spacer peptides, SP1 and SP2. After cleavage, HIV-1 undergoes a dramatic structural rearrangement resulting in the condensation of the conical capsid core and an infectious virus. Viral maturation is a prerequisite for infectivity and has therefore become an attractive target for therapeutic intervention. Atomic-level understanding of the viral maturation mechanism, including critical structural and dynamics details of maturation intermediates and their interactions with maturation inhibitors, is currently lacking. In this dissertation, assemblies of HIV-1 maturation intermediates are studied by solid-state magic angle spinning NMR spectroscopy. Using this methodology, insights are provided for intermediates representing both early and late stages of maturation. While monitoring interactions between protein assemblies and maturation inhibitors remains challenging, the characterization of maturation intermediates in the assembled state provides an initial framework for future studies aimed to understand viral maturation in its entirety and the viral inhibition mechanism. NMR is an inherently insensitive technique, and this is a major challenge encountered when studying large protein assemblies by solid-state MAS NMR. Herein, the development and implementation of nonuniform sampling (NUS) methods and alternative data processing protocols are presented. Through the use of exponentially biased NUS schedules considerable inherent sensitivity enhancements on the order of 1.5-2.0 fold per indirect dimension can be obtained without sacrificing resolution. These enhancements were quantified in the frequency domain through the maximum entropy interpolation (MINT) processing, which allows for a linear transformation between the time and frequency domain, even in datasets possessing a high dynamic range. These findings are demonstrated for a variety of biological solids and have enabled the characterization of systems that are limited by their sensitivity.University of Delaware, Department of Chemistry and BiochemistryPh.D