Mass spectrometry based methods for studies of component assembly and structure/dynamics of thiamin diphosphate dependent enzymes

Jieyu Zhou
For the metabolism of a cell, most of the proteins function as complexes rather than by themselves. Understanding not only how proteins behave in isolation but also how they recognize their binding partners is therefore critical for understanding the functions of proteins. In recent decades, our understanding of protein-protein or protein-ligand interactions have moved beyond rigid binding to conformational changes upon binding, multistep ordered assembly, and structural fluctuations occurring
more » ... ithin fully assembled complexes. In this thesis, the Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) and Chemical Cross-Linking Mass Spectrometry (CX-MS) methods were employed to study the assembly of human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc). Our experiments revealed the binding loci, which is essential to the assembly of human OGDHc. Our findings provide a new insight to understand the structure, organization and assembly of the human OGDHc. Moreover, we have used HDX-MS to explore the protein-ligand interactions of 1-deoxy-D-xylulose 5-phosphate synthase (DXPS). The unique peptides exhibiting EX1-type HDX kinetics found in this thiamin diphosphate (ThDP) dependent enzyme enable us to understand how DXPS structure and dynamics respond to different ligands. A better picture of ligand induced DXPS conformational changes can be provided to help elucidate the mechanism of catalysis, and enable structure-based inhibitor design.
doi:10.7282/t3fb559k fatcat:adad5dovbfbf3jchvdaq4dtdoa