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K S Matthews

Publications and source records attributed to K S Matthews.

16 recordsLinked to original sources
PubMed1979

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.

Cysteine↗
PubMed1979

Activity changes in lac repressor with cysteine oxidation.

The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.

Amino Acids↗
PubMed1979

Tryptic core protein of lactose repressor binds operator DNA.

The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.

Amino Acids↗
PubMed1977

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.

Bacterial Proteins↗
PubMed1977

Selectively deuterated amino acid analogues. Synthesis, incorporation into proteins and NMR properties.

Selectively deuterated analogues of histidine, tyrosine, phenylalanine and tryptophan have been synthesized by chemical exchange. These analogues have been characterized by NMR spectrometry and used for growth of bacteria. Active lactose repressor protein has been isolated from cells grown on the deuterated amino acids, and denatured 1H and 2H NMR spectra have been determined for the protein.

Amino Acids↗
PubMed1976

Kinetic studies of inducer binding to lactose repressor protein.

The kinetics of binding of the inducer, isopropyl-beta, D-thiogalactoside to lactose repressor from Escherichia coli was studied by stopped flow rapid mixing techniques. Three different spectral probes for measuring changes in the conformation of the repressor were used: ultraviolet absorbance, fluorescence, and a reporter group, 2-mercuri-4-nitrophenol, in the visible region. Repressor can be reacted with this mercurial to modify two of the three free sulfhydryl groups per monomer without loss of inducer or operator binding activities. The observed first order rate constant for the reaction of repressor with 2-chloromercuri-4-nitrophenol at pH 7.5 and 20 degrees was found to be on the order of 0.1 s-1, an unexpectedly slow rate for this type of reaction. Once bound to repressor, the nitrophenol serves as a chromophoric probe to monitor changes in the surrounding environment. The binding of inducer to repressor causes a change in the absorbance of the bound 2-mercuri-4-nitrophenol moiety and exhibits a second order rate of 3.2 x 10(4) M-1 s-1. Similar rates were obtained when binding of inducer is monitored by changes in either the ultraviolet absorbance of fluorescence of tryptophan residues. Since the same rate of spectral perturbation is observed for different regions of the primary structure of the protein, the conformational change produced in response to inducer binding appears to be translated rapidly throughout the protein molecule.

Bacterial Proteins↗