Role of Asp274 in lac repressor: diminished sugar binding and altered conformational effects in mutants.
The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site. Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M). Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions. The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins. Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor. From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding.
Chang
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WI
Department of Biochemistry & Cell Biology, Rice University, Houston, Texas 77251, USA.
Matthews
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KS
D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D008024:Ligands; D012097:Repressor Proteins; D001224:Aspartic Acid; D003545:Cysteine