ROLE OF ASP(274) IN LAC REPRESSOR - DIMINISHED SUGAR BINDING AND ALTERED CONFORMATIONAL EFFECTS IN MUTANTS

The role of Asp(274) in inducer binding of lac repressor has been expl ored by spectroscopic measurements, fluorescence quenching, in vitro i nduction assays, and chemical modification of mutants with conservativ e substitutions at this site. Although no fluorescence emission shift ok characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, an d chemical modification results indicate indirectly that the mutants A sp(274)-->Asn and Asp(274)-->Glu bind sugar, albeit with very low affi nity (>0.1 M). Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding act ivity of these two mutant proteins is altered at very high IPTG concen tration, although in opposite directions. The distinct effects of indu cer on operator binding in these two mutant proteins as well as substa ntial differences in the effect of sugar ligand on chemical modificati on of Cys(107) and Cys(140) by 2-(bromoacetamido)-4-nitrophenol sugges t that the conformation of the protein before and after association wi th sugar may differ in these mutant proteins. Fluorescence quenching a ssays of lac mutant proteins at Asp(274) indicate the proximity of Trp (220) to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the cr ystallographic structure of the homologous purine repressor. From thes e results, we conclude that Asp(274) is in the inducer binding site, t hat the character of this residue is crucial to inducer binding, and t hat interaction of sugar with the side chain at this position may be a ssociated with the conformational change necessary for generating high affinity ligand binding.