RELATING STRUCTURE/FUNCTION IN E. COLI PENICILLIN-PBPS

Summary

Principal Investigator: Robert Rosenberg
Abstract: The objective of this proposal is to learn about the structures of the essential penicillin-binding proteins (PBPs) of Escherichia coli and to understand how the structures of these proteins relate to and affect their function in the synthesis of the cell wall peptidoglycan. Many questions still exist as to how these PBPs control such diverse processes as elongation, determination of cell shape,and cell division. Since PBPs are killing targets for B- lactam antibiotics, the need to learn more about their functions provides a compelling reason for the continuing study of these proteins. An understanding of the molecular basis of B-lactam recognition and peptidoglycan biosynthesis should contribute greatly to the rational design of new antibiotics. The specific aims of this proposal are: 1) to continue to supply protein for the solving of the crystal structure of a soluble form of a mutant PBP 5 (in collaboration with Dr. Brian Sutton, Kings College, London), 2) to construct expression systems for the overproduction and eventual crystallization of soluble PBPs 1A and 1B, 3) to construct chimeric proteins of PBPs 1A, 1B, 2 and 3 by fusing the transpeptidase (B-lactam-binding) domain of one PBP with the transglycosylase domain of another PBP, and use these chimeras to study the functions and interactions of the different domains by assaying their affects on cell morphology and viability, 4) to produce B-lactam-binding domains from any of the high molecular weight PBPs and use them for crystallographic studies as well as investigating the domain structures of these PBPs, and 5) to exchange the cytoplasmic and transmembrane regions of PBPs 1B, 2, and 3 with each other and assaying whether these regions have any function in the regulation of activity of the PBP. These experiments combine the techniques of molecular biology with those of biochemistry and crystallography. With these approaches, it is hoped that much useful information will be gained on the structural aspects of PBPs and how these structures relate to their observed function.
Funding Period: 1988-12-01 - 1994-11-30
more information: NIH RePORT