Xuong Nguyen-Huu
e-mail: nxuong@ucsd.edu |
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Our laboratory pursues two directions of research on protein crystallography. The first direction is to improve the methodology, including making new instruments. We are now working hard on designing and building a new X-ray area detector that will allow taking a sequence of up to 16 time-resolved laue pictures, each lasting about 10 millisecond. This method will allow us to follow the structural changes of an enzyme during a reaction. This work will include state-of-the-art engineering and detector physics as well as new computer hardware and software.
The second direction is to use existing protein crystallography methods to solve 3D structures of new and interesting proteins. Following are the list of enzymes being studied in our laboratory.
1. STRUCTURE OF THE CAMP-DEPENDENT PROTEIN KINASE. (IN COLLABORATION
WITH DR. S. TAYLOR, DEPT. OF CHEMISTRY & BIOCHEMISTRY, UCSD).
Our long term goals are to understand the structure
and function of the catalytic (C) and regulatory subunits of cAMP-dependent
protein kinase (PKA). By probing this simple protein kinase, we also
hope to elucidate many of the general rules for this large family of
related enzymes that play critical roles in signal transduction. We
have succeeded in solving both the catalytic (C) and regulatory (R)
units separately. We have now obtained crystals of the RC complex and
are in the process of
solving its structure. In addition, we have solved many structures of
the C subunit complexed with Balanol, a high affinity natural product
inhibitor of PKA and PKC, and with other ATP analogs and peptide inhibitors.
These structures represented both closed and open conformations of the
enzyme and allow us to evaluate its conformational flexibility.
2. STRUCTURE OF A COMPONENT (SPOOF) OF THE SPORULATION SIGNAL TRANSDUCTION
PATHWAY. (IN COLLABORATION WITH DR. JIM HOCH, THE SCRIPPS RESEARCH INSTITUTE.)
The overall aim of this research is to study
the structure and function of a main component (SpoOF) of the sporulation
signal transduction pathway in Bacillus subtilis by X-ray crystallography
and site directed mutagenesis. We have crystallized and solved the structures
of the wild SpoOF and of its Y13S mutant which is resistant to phosphatase
activity by SpoOL. It is an alpha/beta protein with a central beta-sheet
consisting of five strands surrounded by five alpha helices. The folding
of the
structure is similar to CheY, the only other known response regulatory
protein structure; however the comparison of the structures show that
there are significant differences at the C terminal. In the structure
of the mutant, a Ca(2+) ion is bound in the active site. This cation
is missing in the wild type structure. A comparison of the 2 structures
reveals that the cation induces significant changes in the active site.
We are trying to crystallize and solve the
structure of other components of this interesting sporulation signal
transduction pathway.
Datte, P., Beuville, E., Beche, J.-F., Cork, C., Earnest, T., Millaud, J., Nygren, D., Padimore, H., Turko, B. and Xuong, N.-H. (1997). A Prototype 8x9 Pixel Array X-ray Detector for Protein Crystallography. Nucl. Instr. & Methods in Phys. Res. A 391:471-480.
Narayana, N., Cox, S., Xuong, N.-H., Teneyck, L.F. and Taylor, S.S. (1997). A Binary Complex of the Catalytic Subunit of cAMP-Dependent Protein Kinase and Adenosine Further Defines Conformational Flexibility. Structure 5:921-935.
Madhusudan, Zapf, J., Hoch, J.A., Whiteley, J.M., Xuong, N.-H. and Varughese, K.I. (1997). A Response Regulatory Protein with the Site of Phosphorylation Blocked by an Arginine Interaction: Crystal Structure of SpoOF from Bacillus subtilis. Biochemistry 36:12739-12745.
Su, Y., Dostmann, W.R.G., Herberg, F.W., Durick, K., Xuong, N.-H., Ten Eyck, L., Taylor, S.S. and Varughese, K.I. (1995). Regulatory Subunit of Protein Kinase A: Structure of Deletion Mutant with cAMP Binding Domains. Science 269:807.
Knighton, D.R., Zheng, J., Ten Eyck,
L.F., Ashford, V., Xuong, N.-H., Taylor, S.S. and Sowadski, J.M. (1991).
Crystal Structure of the Catalytic Subunit of Cyclic Adenosine Monophosphate-Dependent
Protein Kinase. Science 253:407-414.
Xuong Nguyen-Huu received his Ph.D. from UC Berkeley. His awards include a Guggenheim fellowship, a NATO Senior Fellowship, and a UCSD Chancellor's Associates Award.