Structural studies of reassembled and intact thioredoxin by high-resolution solid-state NMR magic angle spinning spectroscopy.

摘要

Understanding the fundamental roles that proteins play in living organisms is one of the most fascinating and intensive areas of research. Proteins perform a variety of physiological processes that are vital for survival. However, the malfunctioning of proteins can also lead to serious and even fatal diseases. Protein function is closely related to their three-dimensional structure. Solution NMR and X-ray crystallography have greatly contributed to the better understanding of protein structure and function playing an important role in developing structure-based therapeutic drugs. Nevertheless, these structural techniques are restricted to proteins that are either soluble or crystalline, leaving out the possibilities to explore detailed structural information for many proteins of biological relevance that do not fall in those categories. Solid-state NMR is a complementary and emerging approach that provides structural information at atomic-level resolution of proteins that are not soluble or crystalline.; The focus of this dissertation is establishing novel solid-state NMR methodologies to probe three-dimensional structures of proteins, protein interfaces and protein assemblies at atomic-level resolution. Using uniformly and differentially enriched E. coli thioredoxin and its reassembly as an example, we have developed protocols for resonance assignments, secondary structure determination and identification of tertiary constraints via multidimensional magic angle spinning solid-state NMR methods. The experimental solid-state NMR protocols established in this work are expected to be applicable to structural studies of other significantly larger and more complex systems intractable by other structural methods.