Atomic-Level Dynamical, Structural and Functional Investigation of a Membrane Protein Complex through Nuclear Magnetic Resonance Spectroscopy.

Abstract

Biological cell membranes are vital boundaries that separate the intracellular elements from the extracellular environments, and they are fundamental regulators of a number of cellular and physiological phenomena. In fact, these biological processes are essentially based on biomolecular interactions within and between cells. A significant number of biologically important protein-protein and protein-lipid interactions in life science, for instance, signal transductions (an essential molecular machinery for sensory systems), electron transport chains (an essential scheme for respiration systems) and photosynthesis (one of our primary sources of energy), all take place at the membrane interface of cells. Furthermore, more than 30% of the human genome and 50% of known drug targets are membrane-associated proteins. Therefore, it is critical to establish techniques that will allow us to investigate these kinds of membrane complex systems and gain insights into biological phenomena for scientific and biomedical purposes. Despite their importance there are very few reports on the atomic-level structure and dynamics investigations of the combinatorial complexes composed of protein-protein interactions and protein-lipid interactions within bilayers. The lack of success in this area of research is largely attributed to the challenges imposed by membrane proteins when examined with the most commonly used biophysical techniques such as X-ray crystallography, electron microscopy and Nuclear Magnetic Resonance (NMR) spectroscopy. My dissertation concentrates on three novel components that will accelerate the understanding of these important and challenging systems. The first element is to gain atomic-level structure and dynamics determination of biologically important systems by developing new solid-state and solution NMR approaches that enhance resolution and the sensitivity of spectra, allowing for the robust NMR experiments for samples that have higher molecular weight. The second component pertains to the preparation of stable and well-behaved biologically relevant samples. The third is to retain the biological functions of membrane complex systems in order to investigate dynamical structure of membrane complexes. The combination of these three approaches has already led, for the first time, to the determination of structural and dynamical interactions in an intact mammalian membrane protein complex, rabbit Cytochrome-b5, and Cytochrome-P450, which metabolizes more than 50% of the pharmaceuticals in clinical use today.