The Phosphoenolpyruvate:glycose phosphotransferase system (PTS) protein IIIGlc was investigated by nuclear magnetic resonance (NMR) techniques in order to better understand the function of the protein. Studies were carried out to characterize (1) the three-dimensional (3D) molecular and chemical structure of phosphorylated IIIGlc (P-IIIGlc) and the interactions of IIIGlc with the PTS phosphocarrier protein HPr. Phosphorylation of IIIGlc affects binding of the protein to sugar permeases, and thereby regulates the uptake of specific sugars by the cell. A regeneration system was developed that stabilized P-IIIGlc for many days, making it possible to show that the structure of P-IIIGlc was essentially identical to that of IIIGlc. Hence, a change in protein electrostatics rather than structure is responsible for the different activities of IIIGlc and P-IIIGlc. In addition, the chemistry of the active site histidine residues was characterized in detail for IIIGlc and P-IIIGlc. The structure and active site chemistry were also elucidated for the H90Q variants of IIIGlc and P-IIIGlc. The results suggested that the interaction of H75 and H90 in P-IIIGlc facilitates phospho transfer. Finally, we have followed the interaction of IIIGlc and P-IIIGlc with HPr by observing the changes in chemical shift of IIIGlc amide groups that resulted when increasing levels of HPr were added to a solution of IIIGlc. Although the interactions between the proteins appear to be weak, they nonetheless involve specific amino acid residues at the active sites of both proteins. The significance of this project lies in its potential for providing a rational quantitative understanding of the function of the PTS. The PTS has essential and diverse physiological roles in many bacterial cells, including those responsible for dental caries.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Intramural Research (Z01)
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National Institute of Dental & Craniofacial Research
United States
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