X-RAY STUDIES OF CHEY--CHEMOTAXIS PROTEIN FROM E. COLI

本研究计划的基本目标是

The fundamental objective of this research proposal is to investigate the structure and function of a principal chemotaxis control protein from E. coli, on a molecular level. Specifically, the aim is to determine the three-dimensional molecular structure of the CheY protein by x-ray diffraction methods, and to relate the structure of its normal and mutant forms to its function in the control system of bacterial chemotaxis. The bacterial chemotaxis system is an ideal model of studying the molecular control of a cellular behavioral response. A detailed understanding of the system's organization now exists on the genetic level; to explain the control system of chemotaxis on a molecular basis, knowledge of the three-dimensional structures of its protein components is essential. The cheY gene product, because of its central role in the chemotaxis signal processing system, has been chosen as the first component to be analyzed by x-ray diffraction. An additional importance in solving the three- dimensional structure of CheY lies in its primary sequence homology with seven other proteins, all regulators in a variety of cellular functions. These homologies make CheY representative of a new type of regulatory domain for an entire class of bacterial proteins. CheY has been isolated and sequenced, and inactive point mutants have also been sequenced, and phenotypically characterized. Reproducible conditions for obtaining large, single crystals of the wild type CheY protein have been established. These crystals diffract to a Bragg spacing beyond 2.7 A. A potential heavy atom derivative of the CheY crystals has also been prepared. Within the proposed project period, the three-dimensional structure of the CheY protein will be solved using both the conventional multiple isomorphous replacement methods and the techniques of non-crystallographic symmetry averaging. The molecular structure of CheY will be used with the available genetic results to study the structure-function relationships that form the basis of signal processing in chemotaxis.