Structure-function studies on the leucine-binding proteins of Escherichia coli.

Two leucine-binding periplasmic proteins participate in osmotic-shock sensitive transport of leucine (LS-BP) and leucine, isoleucine, valine, and threonine (LIV-BP) in Escherichia coli. This dissertation focuses on two aspects of the function of these proteins and the structural features that are responsible for each. The contribution of the mature portion of LS-BP to its efficient secretion to the periplasmic space was examined using a series of deletion mutations in LS-BP that was constructed by Bal31 exonuclease treatment of the livK gene. Deletion clones that are missing some of the first thirteen residues of the mature protein were processed from precursor to mature form and secreted more slowly than wild-type LS-BP and other deletion proteins. An E. coli strain carrying a deletion of both leucine-binding protein genes was constructed. Restoration of ($\sp3$H) leucine transport in the resulting strain, AE510, required a binding protein gene plus four genes encoding LIV-I/LS membrane components, confirming the involvement of the membrane components LivM and LivF in transport. It was also determined that strain AE510 is a $\beta$-alanine auxotroph, leading to the designation of the open reading frame located between livJ and livK as balA. Based on the prediction of a putative leucine binding site in the N domain of LIV-BP (Sack, et al., J. Mol. Biol. 206:171, 1989), mutations were introduced in both the livJ and livK genes at codons 18, 100, and 276 to attempt to alter the specificity for branched-chain amino acids. Both wild-type proteins and seven mutants were overexpressed using the T7 RNA polymerase/promoter system. It was shown that LIV-BP is inhibited by leucine, isoleucine, valine, threonine, and allo-isoleucine while LS-BP is inhibited by leucine and 5$\sp\prime$5$\sp\prime$5$\sp\prime$-trifluoroleucine (TFL). Site-directed mutants showed varying degrees of alteration in binding constants for different ligands. Residue 100 contributes to recognition of TFL while position 18 may be involved in stabilizing the 'closed' (bound) conformation of the protein. Taken together, the results suggest that the leucine binding site proposed by Sack, et al. contributes to binding and discrimination among the branched-chain amino acids, but is not likely to be solely responsible for either binding or specificity.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103095/1/9034368.pdfDescription of 9034368.pdf : Restricted to UM users only