Two-component regulatory systems are one of the most common mechanisms for signal transduction in bacteria and have recently been found in eukaryotes. In these systems, a histidine kinase autophosphorylates in response to an envoironmental stimulus, providing the phoshate for the response regulator which, subsequently, transduces the signal to a downstream target. Nothing is known about the structural basis of the activation of response regulators upon phosphorylation because the lifetime of the phosphorylated protein is prohibitively short for structural analysis. We are using the response regulator, NTRC, which controls nitrogen metabolism, as a model system. The structure of the unphosphorylated form has been determined in our lab (Volkman, et al., Biochemistry 34, 1413-1424). We have recently obtained conditions which maintain the phosphorylated state long enough for structure determination by NMR. This is done by creating a steady state equilibrium using a small molecule as a phosphodonor. However, due to aggregation and fast turnover, the protein concentration is limited to 0.5 mM. Therefore, structure determination would be greatly facilitated by the higher sensitivity of a 750 MHz magnet and an 8 mm probe. HSQC's taken in our laboratory indicate that the conformational change upon phosphorylation involves only a portion of the molecule so that the known NMR data on the unphosphorylated form will be helpful in analyzing the activated, phosphorylated form. This work should provide the first example of structural understanding of activation via phosphorylation in the response regulator superfamily. Given the high sequence and structural homology among two-component systems, the structure/function relationships in NTRC should be general for other response regulators. Furthermore, the structural changes of a protein triggered by phosphorylation is of interest because this is one of the most common covalent modifications used for modulation of protein function.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR002301-14
Application #
6281642
Study Section
Project Start
1998-04-01
Project End
1999-02-28
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
14
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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