The long term goal of this project is the characterization of the structure, function, assembly and regulation of Kdp, a K-transport ATPase of Escherichia coli.
Aims for this period are: A. IMPROVE PURIFICATION OF KDP. To obtain high expression in which all kdp structural genes are translated at a high rate we will prepare and test other constructs of the structural genes in expression vectors. Purification methods will be explored further to obtain a scheme to obtain active enzyme in high purity and good yield. B. BIOCHEMICAL STUDIES OF KDP. Basic structural features of the Kdp complex (determine N-terminus of the B subunit, C-termini of all 3 subunits, site of acylphosphorylation, presence of leader peptide in complex) will be determined, as will the detailed kinetic properties of the ATPase. Analysis of mutant complexes and crosslinking studies will be used to establish size of the Kdp complex. C. TOPOLOGIC STUDIES OF KDP. We will begin by examining accessibility of different regions of the complex to proteases and to labeling reagents using the detergent-purified enzyme, and then proceed to similar studies in situ using material froms cells that overexpress Kdp. By examining accessibility in Kaback and in inside-out vesicles and comparing these with accessibility in the soluble complex a partial picture of the structure of Kdp may emerge. D. ANALYSIS OF ALTERED FUNCTION MUTANTS. DNA sequence and transport kinetic analysis of mutants will reduced affinity for K will be pursued. We will extend this analysis to the complicated mutants that also reduce the maximum rate which we have not examined much to date. This work will help tell us where and how Kdp binds K for transport, and may reveal information about subunit interactions. E. REGULATION OF KDP. Under this heading four aspects will be pursued: 1) Complete the DNA sequence of the kdpD and kdpE genes. 2) Analysis of the kdpABC promoter region to determine extent of the promoter and look for protein binding to the promoter. 3) Cellular localization of the KdpD protein and tests for binding of KdpE to KdpD. 4) Determine the function of the hydrophobic leader peptide in regulation.
