STRUCTURE & DYNAMICS OF METAL-CONTAINING PROTEINS BY NMR

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The primary goal of this project is to determine the solution tertiary structure of putidaredoxin (Pdx), a 11.6 kD protein consisting of a single 106-residue polypeptide chain and a 2Fe-2S prosthetic group. The determination will be performed using phase-sensitive two dimensional NMR methods, including direct and relayed coherence transfer 1H experiments, homo- and heteronuclear multiple-quantum, Hartmann-Hahn coherence transfer and NOESY experiments. Pdx has bee cloned and is readily isolated from the bacterial expression system which we employ. NMR samples will be prepared both as natural abundance samples for 1H experiments and isotopically labeled with 13C and 15N either randomly or site-specifically for inverse detection experiments. Knowledge of the solution structure of Pdx is critical for a complete understanding of the mechanism of electron transfer between Pdx and its physiological redox partners, Pdx reductase and P-450cam. However, no structure has yet been assigned by X-ray crystallographic methods because of difficulty in obtaining suitable crystals, and NMR methods offer the best route to a rapid determination of tertiary structure. Pdx acts as both reductant and effector for the ternary complex of P- 450cam, camphor and O2 which results in the 5-exo-hydroxylation of camphor in the first step in camphor catabolism by Pseudomonas. The camphor hydroxylase system has been studied for many years as a model for monooxygenase activity, which is important in myriad biological processes, including hormone biosynthesis and carcinogen activation. In the course of this project, we also will be investigating the mechanism of recognition and incorporation of the 2Fe-2S center into Pdx, determining the solution structure of the apoprotein by NMR and its relationship to the holoprotein and observing the thermodynamics of the transitions between folded and unfolded states in the apo- and holoproteins calorimetrically.