Ultraviolet difference spectra of the lactose repressor protein

Matthews, Kathleen S.; Matthews, Hayden; Thielmann, Heinz Walter; Jardetzky, Oleg

doi:10.1016/0005-2795(73)90083-4

Cited by 39 publications

(12 citation statements)

References 10 publications

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“…The sedimentation procedure described above for measuring repressor-DNA binding constants was devised because the optical changes brought about in the DNA and the repressor as a consequence of the interaction of the components (Matthews et al, 1973;Butler et al, 1977) are too small to be useful for this purpose at the required precision of measurement. Centrifugation separates repressor-DNA complexes from repressor free in solution; the rapid sedimentation of the heavy DNA-protein complexes can be followed (Figure 1), and the concentration of the unbound repressor (Rf) calculated from the absorbance of the slowly sedimenting protein which remains behind.…”

Section: Resultsmentioning

confidence: 99%

Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA

Revzin¹,

Hippel²

1977

Biochemistry

169

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Binding parameters for the interaction of lac repressor with non-operator DNA have been determined using a sedimentation velocity technique. Analytical ultracentrifugation is used to separate DNA-protein complexes from unbound protein, thereby permitting direct optical determination of the concentration of free protein as a function of input DNA and repressor concentrations. The method yields absolute values for the association constant ( K ) for proteins binding nonspecifically to nucleic acid lattices; the binding site size ( n ) , and the binding cooperativity parameter ( w ) can also be estimated by this approach. Values of K for the binding of repressor to non-operator DNA have been determined under a variety of solvent conditions. At 0.15 M Na+, 20 OC, pH 7.5, the average value of K for repressor binding to native phage X DNA is 2.4 X IO5 M-' (DNA concentration in base pairs, protein in repressor tetramers). Binding is very ionic strength dependent; we find that 6 log K/6 log [Na+] N -10. Analysis of the ionic strength dependence by the method of Record, M. T., Jr., Lohman, T. M., and de Haseth, P. ((1976), J . Mol.

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Section: Resultsmentioning

confidence: 99%

Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA

Revzin¹,

Hippel²

1977

Biochemistry

169

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“…Some are inducers and decrease bind- ing affinity; others increase affinity and are known as antiinducers (12). IPTG, an inducer, is known to cause slight conformational changes in the lac repressor (15)(16)(17). Under conditions where specific binding to BrdU-Xh8Odlac DNA is seen, the presence of IPTG during irradiation completely eliminates the formation of stable complex ( Fig.…”

Section: Methodsmentioning

confidence: 99%

Photochemical Attachment of lac Repressor to Bromodeoxyuridine-Substituted lac Operator by Ultraviolet Radiation

Lin

Riggs

1974

Proc. Natl. Acad. Sci. U.S.A.

115

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The transducing phage Xh8Odlac carries the lac operator, whereas wild-type Xh8O does not. We find that in high salt (0.18 M XCI), ultraviolet radiation causes the formation of a very stable complex between repressor and 5-bromodeoxyuridine (BrdU)-substituted Xh8Odlac but not to BrdU-Xh8O DNA. Studies with inducers of the lac operon confirm the specificity of attachment. In low salt (0.01 M KC1), ultraviolet radiation will also attach repressor nonspecifically to BrdU-Xh80 DNA. The stability of the complex suggests that covalent bonds are formed. We also report that another regulatory protein, the catabolite gene activator protein, can be attached similarly to DNA.Smith first noted that ultraviolet (UV) radiation cross-links protein to DNA, both in vivo and in vitro (1). The experimental evidence for cross-linking was that after UV treatment, DNA was not extractable from sodium dodecyl sulfate (SDS)-protein precipitates. This work has been reviewed (2). Proteins known to bind to DNA were not studied. Recently, Markovitz (3) demonstrated that UV irradiation results in covalent bond formation between DNA polymerase and DNA. Stimulated by the work of Markovitz, we tried to demonstrate the specific cross-linking of lac repressor to lac operator in Xh8Odlac DNA. These experiments were not successful until 5-bromodeoxyuridine (BrdU)-substituted Ah80dlac DNA was used. We report here the photochemical attachment of lac repressor specifically to BrdU-substituted lac operator. METHODSWe prepared lac repressor (isUPTrq) from strain M96 following the procedure of Muller-Hill, Beyreuther, and Gilbert (4). To ensure purity, additional chromatography on DEAESephadex was done (5). The preparation was free of impurities detectable by SDS-acrylamide gel electrophoresis and all DNA-binding activity (including photochemical cross-linking) sedimented in a sucrose gradient as lac repressor. The nitrocellulose filter assay for repressor-DNA complexes has been described in detail (6, 7). Because of somewhat lower background and better reproducibility, we are now using type For most experiments, ultraviolet light treatment was at a distance of 11 cm from a short wavelength mineral light (Ultraviolet Products, model UVS-11). The sample (0.75 ml) was in 0.5 X 2-inch polyallomer tubes situated directly below the UV lamp. Irradiation was usually done at room temperature (250) in buffer I, which contains: 10 mM KCl, 10 mM Tris HCl (pH 7.4), 0.1 mM EDTA, 0.1 mM dithiothreitol, 5% (v/v) dimethylsulfoxide, and 50 Ag/ml of BSA. The BSA was heat treated at 700 for 2 hr at pH 9.0. UV dosage was measured with an ultraviolet meter (Ultraviolet Products, model J-225). Test tubes with the bottom cut out were used to estimate the dose actually received by the sample. RESULTS The specific binding of lac repressor to lac operator has been firmly established (10, 11) and studied in detail using nitrocellulose filters to asay for repressor-operator complex (6,(12)(13)(14). Repressor binding to operator is eliminated by isopropyl-B-D-thiogalactoside ...

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“…Previous studies indicated that inserting tryptophan into a protein matrix shifts max , the wavelength of maximal absorbance, by about ϩ4 nm at most, and has negligible effect on the spectral lineshape (McLaren and Shugar, 1964;Matthews et al, 1973). The relative insensitivity of max to environment is in marked contrast to the extremely large protein-induced shifts (up to Ϫ45 nm) in the wavelength of maximal tryptophan fluorescence (Lakowicz, 1983).…”

Section: Effect Of Environment On Tryptophan Absorptionmentioning

confidence: 96%

Modification of Cyclic Nucleotide–Gated Ion Channels by Ultraviolet Light

Middendorf

Aldrich²,

Baylor

2000

The Journal of General Physiology

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We irradiated cyclic nucleotide–gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the α subunit of bovine retinal cyclic nucleotide–gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more “target” tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.

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Ultraviolet difference spectra of the lactose repressor protein

Cited by 39 publications

References 10 publications

Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA

Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA

Photochemical Attachment of lac Repressor to Bromodeoxyuridine-Substituted lac Operator by Ultraviolet Radiation

Modification of Cyclic Nucleotide–Gated Ion Channels by Ultraviolet Light

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