This project is focused on the structure, function, and conformation of an integral membrane proton pump, transhydrogenase. Transhydrogenase (TH) is an essential enzyme of the respiratory system in mitochondria and bacteria. Proton translocation through membrane-intercalated domain II is coupled to hydride transfer between NAD(H) and NADP(H) bound to extramembranous domains I and III. In the forward direction, the proton motive force shifts the equilibrium toward NADPH formation; in the reverse direction, transhydrogenation from NADPH to NAD results in creation of a proton gradient. The structure of the intact enzyme is unknown, and the mechanism for coupling binding energy and conformational change with proton translocation is unsolved. Structural and functional experiments will develop a picture of this complex molecular machine and its catalytic mechanism.
Three Aims address key aspects of TH function: the sites of proton uptake and release in domain Ill, the mechanism of hydride transfer between domains I and III, and the structure and conformation of the intact enzyme.
A fourth Aim addresses the mechanism by applying mutagenesis and functional assays to evaluate the coupling of proton transfer in domain II with conformational and binding events in domain III. The experiments entail protein expression and purification, mutagenesis, enzymatic activity assays, preparation of monodisperse, detergent-solubilized TH, negative stain electron microscopy, crystallization in lipid bilayers, single-particle and 2D crystal electron cryo-microscopy, crystallization of detergent-solubilized TH, NMR of expressed domain III, and x-ray crystallography of expressed domains I and III, individually and in cocrystals. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061545-05
Application #
6824379
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
2000-07-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
5
Fiscal Year
2004
Total Cost
$446,726
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Leung, Josephine H; Schurig-Briccio, Lici A; Yamaguchi, Mutsuo et al. (2015) Structural biology. Division of labor in transhydrogenase by alternating proton translocation and hydride transfer. Science 347:178-81
Zhao, Yonghong; Sun, Ling; Muralidhara, B K et al. (2007) Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4. Biochemistry 46:11559-67
Smith, Brian D; Sanders, Jason L; Porubsky, Patrick R et al. (2007) Structure of the human lung cytochrome P450 2A13. J Biol Chem 282:17306-13
Zhao, Yonghong; White, Mark A; Muralidhara, B K et al. (2006) Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction. J Biol Chem 281:5973-81
Hunsicker-Wang, Laura M; Pacoma, Ronald L; Chen, Ying et al. (2005) A novel cryoprotection scheme for enhancing the diffraction of crystals of recombinant cytochrome ba3 oxidase from Thermus thermophilus. Acta Crystallogr D Biol Crystallogr 61:340-3
Sundaresan, Vidyasankar; Chartron, Justin; Yamaguchi, Mutsuo et al. (2005) Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains. J Mol Biol 346:617-29
Yamaguchi, Mutsuo; Stout, C David (2003) Essential glycine in the proton channel of Escherichia coli transhydrogenase. J Biol Chem 278:45333-9
Sundaresan, Vidyasankar; Yamaguchi, Mutsuo; Chartron, Justin et al. (2003) Conformational change in the NADP(H) binding domain of transhydrogenase defines four states. Biochemistry 42:12143-53
Prasad, G Sridhar; Wahlberg, Marten; Sridhar, Vandana et al. (2002) Crystal structures of transhydrogenase domain I with and without bound NADH. Biochemistry 41:12745-54
Yamaguchi, Mutsuo; Stout, C David; Hatefi, Youssef (2002) The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli. J Biol Chem 277:33670-5