The overall objective of the research is to elucidate the structural and mechanistic characteristics of microsomal glutathione transferase, a membrane-bound enzyme involved in the catalytic addition of glutathione to toxic reaction intermediates. Very little is known about the structure and mechanism of microsomal GSH transferase (MGST1) other than a low-resolution three-dimensional protein map, which does not address activation of the enzyme and how the protein dynamics may change in response to ligands binding. The research to be undertaken will investigate the role of protein dynamics in the catalytic mechanism of MGSTI. The kinetics of backbone amide HID exchange is well-established as a sensitive indicator of protein structure and dynamics. Amide H/D exchange mass spectrometry (ESI-TOF) provides an amenable technique to study the dynamics of MGST1, with future application to other membrane proteins. The kinetic results should be a valuable tool of assessing the accuracy of the low resolution structure, as well as understanding specific segmental motions that may affect catalysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32ES013105-02
Application #
6849225
Study Section
Special Emphasis Panel (ZRG1-F04 (20))
Program Officer
Humble, Michael C
Project Start
2004-02-01
Project End
2006-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
2
Fiscal Year
2005
Total Cost
$41,608
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Prage, Edward B; Pawelzik, Sven-Christian; Busenlehner, Laura S et al. (2011) Location of inhibitor binding sites in the human inducible prostaglandin E synthase, MPGES1. Biochemistry 50:7684-93
Busenlehner, Laura S; Branden, Gisela; Namslauer, Ida et al. (2008) Structural elements involved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen-deuterium exchange of the E286H mutant. Biochemistry 47:73-83
Busenlehner, Laura S; Alander, Johan; Jegerscohld, Caroline et al. (2007) Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1. Biochemistry 46:2812-22
Busenlehner, Laura S; Salomonsson, Lina; Brzezinski, Peter et al. (2006) Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase. Proc Natl Acad Sci U S A 103:15398-403
Busenlehner, Laura S; Armstrong, Richard N (2005) Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry. Arch Biochem Biophys 433:34-46
Busenlehner, Laura S; Codreanu, Simona G; Holm, Peter J et al. (2004) Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 1. Biochemistry 43:11145-52