Spectroscopic study of Y210C lambda-repressor: implications for cooperative interaction.
A non-cooperative mutant of lambda-repressor, Y210C, has been purified and characterized. The mutant protein does not show any evidence of cooperative interaction as judged by difference near-UV circular dichroism spectra of DNA. The mutant protein also shows much weaker self-assembly as revealed by fluorescence anisotropy measurement. The far-UV circular dichroism spectrum of the protein shows a modest but significant reduction in the 220 nm range, suggesting a structural change. The Lehrer plot of acrylamide quenching of Y210C repressor at a predominantly dimeric concentration (0.5 microM) is almost identical with that of the wild-type protein at the same concentration. Transmission of operator-induced conformational change is also preserved in the mutant protein. Like that of the wild-type protein, cysteines of the mutant protein are unreactive to sulfhydryl reagents under native conditions. Most importantly, C210 is unreactive to sulfhydryl reagents under native conditions. This fact, coupled with the structural change observed in the far-UV CD spectra, suggests that C210 is located at the interior of the protein and exerts its effect indirectly on cooperative contact probably through destabilization of a reverse turn, of which it is an important part.
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Department of Biophysics, Bose Institute, Calcutta, India.
Bandyopadhyay
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Roy
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D010582:Bacteriophage lambda; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D005454:Fluorescence Polarization; D046911:Macromolecular Substances; D008958:Models, Molecular; D016296:Mutagenesis; D009876:Operon; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D013439:Sulfhydryl Reagents; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; D004228:Dithionitrobenzoic Acid; D003545:Cysteine