Structural studies will be carried out for two proteins of the E. coli phosphotransferase system, glycerol kinase and factor IIIglc. The long term goal is to determine the three-dimensional structure of the proteins alone and complexed together in biologically relevant forms. The structural studies will provide the basis for the investigation of the enzymatic mechanism of glycerol kinase, the structural basis for the allosteric regulation of the activity and the nature of the protein-protein complex, which is a phosphorylation dependent signal transduction complex. Four exceptionally high quality crystal forms have been obtained of glycerol kinase in the presence of substrates, products and allosteric effectors. Two useful crystal forms have been obtained of the complex between glycerol kinase and factor IIIglc, and good crystals have been obtained of factor IIIglc alone. The standard procedures of multiple isomorphous replacement, molecular replacement and noncrystallographic symmetry averaging will be used to solve the structures of these crystal forms. The specific questions which will be addressed are: What is the structural basis for the enzymatic activity and velocity modulation of glycerol kinase? Do different allosteric effectors of the activity modulate the structure in similar or different ways? What is the nature of a phosphorylation dependent protein-protein interaction? Does glycerol kinase have structural features in common with other ATPases?
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