Production and X-ray crystallographic analysis of fully deuterated cytochrome P450cam

Abstract

Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 A. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O(2) via a mechanism that is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 A, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.

Production and characterization of recombinant perdeuterated cholesterol oxidase

“…In addition, the final yield of pure perdeuterated protein was approximately 1.5 to 2.0 times lower than that of the hydrogenated protein. The decreased yield is similar to that reported for production of perdeuterated cytochrome P450cam, which also displayed a 2-to 3-fold decrease in protein expression [26], and haloalkane dehalogenase, which had a 1.5-fold decrease in protein yield [25]. Conversely, a similar yield of perdeuterated and hydrogenated protein was obtained for human arginase I [39].…”

“…Perdeuteration may affect the expression and purification of proteins because replacing all 1 H atoms for the heavier 2 H isotope in the growth medium can affect the level of expression of proteins in the cell [25,26]. Expression and purification procedures, therefore, must be optimized for perdeuterated proteins to ensure adequate production of highly pure protein.…”

Substrate binding induces structural changes in cytochrome P450cam

“…These crystallographic results explain the change in spin state and entropy-driven substrate binding (Griffin & Peterson, 1972). Recent X-ray structures of the substratebound form at 1.4-1.6 Å resolution (Schlichting et al, 2000;Meilleur et al, 2005) demonstrate that Thr101 forms a hydrogen bond to the haem 6-propionate, which differs from the previously observed conformation (Poulos et al, 1987). The formation of a hydrogen bond between Thr101 and the haem 6-propionate is important because it raises the redox potential of the haem iron.…”

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

“…With the benefits of deuteration becoming increasingly realizable, more research activities will be dedicated into this direction in the next few years S660 Review. Interfacial assembly of proteins X. Zhao et al (Meilleur et al 2005;Budayova-Spano et al 2006;Di-Costanzo et al 2007;Liu et al 2007;Laux et al 2008).…”