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J A Gardner

Publications and source records attributed to J A Gardner.

At least 19 recordsLinked to original sources
PubMed1991

Energy transfer in lactose repressor protein modified with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate.

Energy transfer between the two tryptophan residues in the lactose repressor protein and the fluorescent moiety of the cysteine-specific reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (1,5-IAEDANS) has been examined. Modification of repressor with this compound did not affect operator or inducer binding. 1,5-IAEDANS reacted primarily with Cys140 in wild-type repressor [Schneider et al. (1984) Biochemistry 23, 2221]; in the presence of inducer, modification at Cys107 increased, while reaction at Cys140 remained unchanged. Energy transfer between tryptophans and the AEDANS moiety(ies) in wild-type lac repressor occurred with an efficiency of 6.7 +/- 1.9% in the absence and 7.8 +/- 1.6% in the presence of inducer. The distance between the Trp donor(s) and the acceptor in wild-type repressor was calculated to be in the range approximately 35 A under both conditions. The similarity in efficiency despite large differences in the amount of acceptor attached to Cys107 when inducer is bound indicates that the AEDANS group at position 107 does not participate significantly in energy transfer and that the label at position 140 acts as the primary acceptor group. The similarity of energy-transfer efficiency (7.1 +/- 3.8%) observed for 1,5-IAEDANS-modified monomeric mutant repressor (Y282D) indicates that the transfer is primarily intrasubunit in the native tetramer. Measurements using two mutant repressors (each with a single tryptophan and modified with 1,5-IAEDANS) demonstrated that both tryptophans can serve as donor in the energy-transfer process. The W201Y repressor (containing Trp220) exhibited a transfer efficiency lower than wild type (5.6 +/- 2.4%), corresponding to a slightly larger distance between the donor-acceptor pair in this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1990

Characterization of two mutant lactose repressor proteins containing single tryptophans.

Two mutant lactose repressors, each containing a single tryptophan, were generated by site-specific mutagenesis. Tyrosine was substituted for tryptophan to be analogous to amber suppression mutants reported previously (Sommer, H., Lu, P., and Miller, J. H. (1976) J. Biol. Chem. 251, 3774-3779). Unlike the amber suppression mutants, plasmids containing the mutant sequences produce large quantities of stable, easily isolable protein. The binding properties of the site-specific mutant repressors (W201Y, W220Y) differ from those reported for the corresponding suppression mutants (A201, A220). Whereas minimal effects on operator dissociation rate from lambda plac DNA were noted for the suppression mutants, purified W201Y and W220Y proteins exhibit 10- and 5-fold reduced affinity for a 40-base pair operator, respectively, compared with wild-type. Inducer binding of the A201 and W201Y mutants was similar to that for wild-type repressor, but the inducer affinity of W220Y was approximately 2-fold lower than A220 (approximately 30-fold lower than wild-type). Fluorescence spectra and iodide quenching of the mutant proteins were similar to the suppression mutants, but the absorption coefficient differed significantly from the values reported previously. Acrylamide and iodide quenching results indicate that Trp201 is relatively buried whereas Trp220 is exposed to solvent; inducer binding reduces quenching of Trp220 significantly. CD spectra indicate that the mutant proteins have secondary structural features similar to those of wild-type. Inducer UV difference spectra showed that the major features reported for the wild-type isopropyl beta-D-thiogalactopyranoside difference spectrum were attributable to both tryptophans. In the presence of melibiose, a new minimum appeared in the difference spectra of wild-type and W201Y which was not evident when these proteins bound isopropyl beta-D-thiogalactopyranoside. It is possible that this new feature results from Trp220 involvement in a direct contact with the second sugar in disaccharide inducer molecules such as melibiose and 1,6-allolactose.

Circular Dichroism↗
PubMed1990

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.

Ligands↗
PubMed1987

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol.

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.

Acetamides↗
PubMed1987

31P NMR studies on recovery from hypoxia of human tumor cells.

We describe the use of 31P NMR spectroscopy in the study of metabolic changes related to hypoxia in cultured human tumor cells in vitro. The 31P NMR spectrum can easily distinguish between metabolically active cells, metabolically inactive "dormant" cells, and necrotic cells. A crucial observation was that of the ability of the "dormant" cells to resume active metabolism on incubation with oxygen after long periods of hypoxia.

Adenocarcinoma↗