Structure Elucidation Of Thioredoxin Reductase By NMR

Understanding the function of a bio-macromolecule requires knowledge of its structure. NMR is an effective method to determine three-dimensional structure of bio-macromolecule at near-physiological conditions. Therefore, biological molecular nuclear magnetic resonance (NMR) draws more and more attention today, a variety of NMR methods have been developed to clarify the structure of biological macromolecular such as protein and DNA molecules in the solution.NMR spectroscopy is widely applied to structure determination of proteins that are the most versatile macromolecules in living systems and participate in a wide range of essential biological processes. In the thesis, the structure of an important oxidation-reduction enzyme-thioredoxin reductase (TrxR) have been elucidation using NMR, in order to understand the function of it.TrxR is a kind of NADPH dependent enzyme which contains FAD structure domain, and belongs to the pyridine nucleotide-disulfide redox enzyme family system, there are two subunits exists in TrxR, NADPH and FAD are all function related. In solution, these two areas will rotation in the process of its biological functions. Therefore, NMR based structure elucidation of TrxR is difficult, because of it unstable strcture and big molecular weight. The thesis mainly focused on how to solve these difficulties:(1) In order to characterize the structure of TrxR, we explored thioredoxin reductase in solution using nuclear magnetic resonance (NMR). There are multiuple conformations of TrxR in solution states, to simplify it, we have to find some methods to control its structure first. Many efforts have been made, such as mutation, structure modification, and structure modulation using agents, etc. Finally, by deuteration, we have collected a series of2D and3D NMR spectra of TrxR, and have had parts of its signals assignmented.(2) NMR signals of the water are much stronger than those of biological solute, which is one of the main constraints to biological NMR, many methods have been developed for solvent suppression. In order to chose the proper solvent suppression method for biological samples we interested, we tested nearlly all important solvent suppression methods with different types of biological solutions, and found out that each of the methods have their special advantages and disadvantages, and should be chosen carefully according the the properties of the samples.