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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM022323-14
Application #
3271085
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-09-01
Project End
1993-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
14
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Buurman, Ed T; McLaggan, Debbie; Naprstek, Josef et al. (2004) Multiple paths for nonphysiological transport of K+ in Escherichia coli. J Bacteriol 186:4238-45
Brandon, L; Dorus, S; Epstein, W et al. (2000) Modulation of KdpD phosphatase implicated in the physiological expression of the kdp ATPase of Escherichia coli. Mol Microbiol 38:1086-92
Gassel, M; Siebers, A; Epstein, W et al. (1998) Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli. Biochim Biophys Acta 1415:77-84
Buurman, E T; Kim, K T; Epstein, W (1995) Genetic evidence for two sequentially occupied K+ binding sites in the Kdp transport ATPase. J Biol Chem 270:6678-85
Altendorf, K; Siebers, A; Epstein, W (1992) The KDP ATPase of Escherichia coli. Ann N Y Acad Sci 671:228-43
Polarek, J W; Williams, G; Epstein, W (1992) The products of the kdpDE operon are required for expression of the Kdp ATPase of Escherichia coli. J Bacteriol 174:2145-51
Walderhaug, M O; Polarek, J W; Voelkner, P et al. (1992) KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. J Bacteriol 174:2152-9
Epstein, W (1992) Kdp, a bacterial P-type ATPase whose expression and activity are regulated by turgor pressure. Acta Physiol Scand Suppl 607:193-9
Epstein, W; Walderhaug, M O; Polarek, J W et al. (1990) The bacterial Kdp K(+)-ATPase and its relation to other transport ATPases, such as the Na+/K(+)- and Ca2(+)-ATPases in higher organisms. Philos Trans R Soc Lond B Biol Sci 326:479-86;discussion 486-7
Walderhaug, M O; Litwack, E D; Epstein, W (1989) Wide distribution of homologs of Escherichia coli Kdp K+-ATPase among gram-negative bacteria. J Bacteriol 171:1192-5