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Spectral studies of lactose repressor protein modified with nitro phenol reporter groups


Journal of Biological Chemistry 252(10): 3153-3159
Spectral studies of lactose repressor protein modified with nitro phenol reporter groups
The spectral characteristics of lactose repressor protein [from Escherichia coli] modified with 2 nitrophenol reporter groups were determined. Reaction of 2-chloromercuri-4-nitrophenol with repressor increases the apparent pK of the nitrophenol from 6.75 to 8.3 and alters the wavelength of maximum absorption. Subsequent binding of inducer shifts the apparent pK toward slightly more acid pH. The pK values of the nitrophenol groups introduced into the repressor by reaction with 2-bromoacetamido-4-nitrophenol could not be determined, since they are below pH 7.0 where repressor is unstable. Titration of the nitrophenol chromophores indicates that the protein structure may undergo transitions as a function of pH. The binding of inducer to the repressor protein results in conformational changes which are reflected in spectral alterations for the nitrophenol groups. The mercurinitrophenols undergo a shift in the wavelength of maximum absorption to shorter wavelength, interpreted as reflecting an environmental alteration which results in more polar surroundings. Conversely, the nitrophenol groups introduced by the alkylating agent have a shift of wavelength of maximum absorption to longer wavelength, apparently experiencing a less polar environment. The positions of the nitrophenol groups in the primary structure of the protein and these spectral observations are correlated. Anti-inducers and a neutral compound also bind to the repressor protein, eliciting similar spectral changes; these alterations are different from those observed for inducers, as predicted from the different effects of these ligands on the function of the protein. The rates of the spectral transitions in response to inducers were measured and were of 2nd order. The rates are similar to those observed previously for other chromophores/fluorophores and apparently reflect a concerted, rapid change in the structure of the protein on binding to inducers.


Accession: 006459932

PMID: 16884



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