MOLECULAR STRUCTURE STUDIES OF BACTERIAL SIGNAL PROTEINS

等位基因特异性抑制是一种常用的遗传方法

Allele-specific suppression is a genetic method commonly used to demonstrate physical interactions among proteins in vivo. Theoretically, mutations identified in suppression experiments define the critical sites of intermolecular recognition in the normal biological system. These conclusions are made in the absence of any structural information, but it is obvious that results from suppression analysis have many structural implications. We argue that multiple suppression sites mapped onto the three-dimensional structure of molecule should define its interactive surface, and that the functionality of this surface should be understandable from the chemical nature of the amino acids at the mutant sites and their interchanges. These properties should be verifiable through three-dimensional structural analyses. The long-term objective of this proposal is to establish a rigorous test case for evaluating the structural validity of allele-specific suppression theory. The CheY protein from the bacterial chemotaxis signal transduction pathway provides an excellent system for this purpose. There is an abundance of structural and genetic information concerning CheY's function: the structural of wild-type CheY is known at high resolution, and nine different suppressor mutants of CheY have been characterized. The plan is to determine the structures of these nine CheY mutants, and use the structural results to test and extend the principles of suppression theory. This work will also require the structural solution of phosphorylation mutants of CheY in order to determine CheY's normal molecular mechanism of signalling. The methods to be used are the standard techniques of single crystal x-ray diffraction. The specific aims of this research are: 1) to determine the three-dimensional structures of nine CheY allele- specific suppressor mutants, identified as V11M, E27K, S56F, A90V, A90T, V108M, F111V, T112I, and E117K; and 2) to determine the three-dimensional structures of four CheY phosphorylation mutants, namely D13N, G39E, T87I, and A88T. These results will greatly improve our understanding of the function of CheY and CheY homologues in bacterial signal transduction systems. Also, this approach may well become a model for studying protein-protein interactions in other signal transduction pathways.