NMR-based characterization of protein interactions: Studies of P450cam, RSP2, Su e and PMK

Huili Yao, Marquette University


NMR spectroscopy is a universal technique for studying protein structure, function, dynamics, as well as protein interactions with ligands or substrates. In this work, NMR spectroscopy was applied to several proteins (or peptide) in order to better understand their function and/or interactions with substrates or ligands. In Chapter I, a novel protein with unknown function, RSP2 (radial spoke protein 2), was investigating using an NMR STD (saturation transfer difference) strategy, called CF-STD (cofactor fingerprinting with STD). The study suggested the RSP2 is a kinase and it prefers to bind cCMP instead of cAMP and cGMP. In Chapter II, the interaction between P450cam and its substrate, camphor, was studied. 1 H NMR T1 relaxation measurements and an in silico docking study were performed, for the P450cam-camphor complex. The second binding site (or entry site) of P450cam was explored, which was not found in the X-ray crystal structure. However, various lines of evidence are presented to support our surprising result. In Chapter III, the sequential backbone assignment was done for the 22 kDa PMK (phosphomevalonate kinase) protein with multidimensional NMR at high-field (i.e. 600 and 800 MHz with cryoprobe). Although assignments were not completed in this study, most of the triple resonance NMR experiments were completed and partial assignments obtained. Following the backbone assignment, we also studied structure changes of PMK upon the binding of ATP and phosphomevalonate. This dynamics study gives very useful information in terms of enzyme mechanism. Finally, in Chapter IV, the structure of transmembrane peptide Su e (subunit e) from yeast ATP synthase was studied using several kinds of 2D NMR methods and CD (circular dichroism) spectroscopy. The dynamics of Su e peptide was explored, too. Su e was characterized as mainly α-helical and the structure of Su e in a DPC (a detergent) micelle was proposed at last.

Recommended Citation

Yao, Huili, "NMR-based characterization of protein interactions: Studies of P450cam, RSP2, Su e and PMK" (2006). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI3244569.