Structure Analysis of a Transient Protein Complex - NMR, X-ray crystallographic and Mass Spectrometric Studies of the Soluble Domains of the Proton-Pump Transhydrogenase from E. coli.
Interactions between macromolecules in the cell are fundamental to life. However, many interactions are of a transient nature and are as such difficult to study at the atomic level using existing techniques. Therefore, a method extending the use of nuclear magnetic resonance (NMR) spectroscopy and paramagnetic spin labels was developed, with which it is possible to determine the structure of a transient macromolecular complex to a high precision.
This thesis primarily focuses on the characterisation and the structure determination of the complex of the soluble domains of transhydrogenase from Escherichia coli, using NMR spectroscopy. Further studies include the structure determination of the individual domains I and III by X-ray crystallography and NMR spectroscopy, respectively. Initial results of interaction studies using high-resolution mass spectrometry and other NMR techniques are also discussed.
Transhydrogenase is a membrane protein which is present in many species throughout the tree of life. This enzyme uses the electrochemical proton gradient, created by the enzymes in the respiratory chain, to catalyse the generation of NADPH by the oxidation of NADH. Transhydrogenase is therefore located in the interface between three major components of the metabolism: the co-factors NAD(H) and NADP(H), and the electrochemical proton-gradient. Transhydrogenase is known to be an important provider of NADPH, which is used both in biosynthesis and for protection against malignant radicals in the cell.
nuclear magnetic resonance