HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Spotts, R. O. Articles by Matthews, K. S. Search for Related Content PubMed PubMed Citation Articles by Spotts, R. O. Articles by Matthews, K. S. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 266, Issue 34, 22998-23002, 12, 1991

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins

RO Spotts, AE Chakerian and KS Matthews
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Muller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				T. C. Flynn, L. Swint-Kruse, Y. Kong, C. Booth, K. S. Matthews, and J. Ma Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimer Protein Sci., November 1, 2003; 12(11): 2523 - 2541. [Abstract] [Full Text] [PDF]

				M. Schumacher, K. Choi, H Zalkin, and R. Brennan Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices Science, November 4, 1994; 266(5186): 763 - 770. [Abstract] [PDF]