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K S Matthews

Publications and source records attributed to K S Matthews.

At least 55 records · Page 3Linked to original sources
PubMed1993

Model of lactose repressor core based on alignment with sugar-binding proteins is concordant with genetic and chemical data.

Using primary sequence similarity to arabinose-binding protein, D-glucose/D-galactose-binding protein, and ribose-binding protein (Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1991) J. Biol. Chem. 266, 5226-5237; Mowbray, S. L., and Cole, L. B. (1992) J. Mol. Biol. 225, 155-175), the core domain (residues 62-323) of the bacterial regulatory protein lac repressor has been aligned to these sugar-binding proteins of known structure. Although the sequence identity is not striking, there is strong overall homology based on two separate matrix scoring systems (minimum base change per codon (MBC/C) and amino acid homology per residue (AAH/R)) (mean score: MBC/C < 1.25, AAH/R > 5.50; random sequences: MBC/C = 1.45, AAH/R = 4.46). Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar-binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of the lac repressor has been modeled based on the known structures of the sugar-binding proteins as templates. While the structure deduced for the repressor is hypothetical, the model generated allows a comparison between the predicted tertiary arrangement and the wealth of genetic and chemical data elucidated for the repressor. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. This approach, therefore, provides a means to visualize the core domain of the lac repressor that allows interpretation of genetic and chemical data for specific residues and rational design of future experiments.

Amino Acid Sequence↗
PubMed1993

Lysine 84 is at the subunit interface of lac repressor protein.

Mutations have been generated at the Lys84 site of the lac repressor to explore its predicted role in inducer binding and/or subunit interaction. Four single mutations, Lys84-->Ala, Lys84-->Leu, Lys84-->Arg, and Lys84-->Glu, have been generated by site-specific mutagenesis. In addition, the mutation Tyr282-->Asp, which results in a monomeric repressor, has been coupled with these four single mutants to generate the four corresponding double mutants. Unchanged inducer binding affinities in all Lys84 mutants except Lys84-->Arg suggest that Lys84 does not contribute energy to inducer binding and is not found in the inducer-binding site as previously proposed (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). Interestingly, the double mutants with hydrophobic side chains at the Lys84 site are tetramers, while those with charged side chains remain monomers. This result agrees with the recent model of the lac repressor (Nichols, J. C., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612), in which Lys84 is mapped by sequence alignment to the same face of the subunit as Tyr282. More detailed inducer binding, operator binding, and immunoblotting studies show that all the mutations at Lys84 have quaternary structures that deviate from wild-type protein, providing supportive evidence for the model placing this residue on the surface of the monomer subunit. Substitution of Lys84 by Ala or Leu results in 100-200-fold decreased association and dissociation rate constants for inducer binding and biphasic character. This decrease can be rescued at least partially in the respective double mutants at elevated pH, at which wild-type repressor shows a 10-fold decrease in affinity and cooperativity in inducer binding. In all substitutions with Ala or Leu, immunoblotting patterns with monoclonal antibody, an assay sensitive to alterations in quaternary structure, are distinct from wild-type repressor. Although substitution with Arg at position 84 yields a protein with 10-fold lower inducer binding affinity, the mutant shows decreased pH dependence of inducer binding. Substitution at this site with Glu results in cooperativity at neutral pH with no change in inducer binding at elevated pH. In addition, operator binding affinity of this mutant is affected by elevated pH, a phenomenon not observed in wild-type repressor. These changes in inducer and operator binding properties appear to be related to the altered quaternary structure of these mutants at Lys84.

Chromatography, Gel↗
PubMed1992

Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation.

The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., and Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Krämer, H., and Müller-Hill, B. (1991) New Biol. 3, 57-62). To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed. The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor. The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography. Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins. Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion. Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH. Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer. "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed. The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored. This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure.

Amino Acid Sequence↗
PubMed1992

T41 mutation in lac repressor is Tyr282----Asp.

A monomeric mutant of the lac repressor protein, designated T41, produced originally by the in vivo MutT mutagenesis exhibited behavior similar to mutants identified as Tyr282----Ser or Tyr282----Gln [Schmitz et al., J. Biol. Chem. 251 (1976) 3359-3366]. The T41 gene, encoded within a phage lambda prophage in the Escherichia coli genome, was amplified by polymerase chain reaction and sequenced. The only mutation found in the nucleotide sequence corresponded to position 282 in the protein, and the amino acid encoded was Asp. The Tyr282----Asp mutant protein produced by site-specific methods was isolated and shares the oligomeric and inducer binding characteristics previously determined for the T41 mutation.

Asparagine↗
PubMed1992

Effect of lac repressor oligomerization on regulatory outcome.

Regulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production. Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription. The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein. The lac operon in Escherichia coli provides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes. DNA loop formation is essential for maximal repression of the lac operon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self-associate to form a bidentate tetramer. The stability of this looped complex is enhanced significantly by DNA supercoiling. Tetramer assembly from dimers apparently occurs via interactions of a 'leucine zipper' motif in the C-terminal domain of the protein, and the tetramer is essential to formation of looped complexes. Furthermore, analysis of the DNA-binding characteristics of dimeric mutants has established that the monomer-dimer association and dimer-DNA binding (monomer does not bind to DNA) are coupled equilibria. Thus, dimer assembly is essential for generating a DNA-binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis. Protein-protein interactions therefore play a pivotal role in the regulatory activities of the lac repressor and must be considered when analysing the activities of any oligomeric DNA-binding protein.

DNA, Bacterial↗
PubMed1992

DNA looping.

DNA-looping mechanisms are part of networks that regulate all aspects of DNA metabolism, including transcription, replication, and recombination. DNA looping is involved in regulation of transcriptional initiation in prokaryotic operons, including ara, gal, lac, and deo, and in phage systems. Similarly, in eukaryotic organisms, the effects of enhancers appear to be mediated at least in part by loop formation, and examples of DNA looping by hormone receptor proteins and developmental regulatory proteins have been found. In addition, instances of looped structures have been found in replication and in recombination in both prokaryotes and eukaryotes. DNA loop formation may have different functions in different cellular contexts; in some cases, the loop itself is requisite for regulation, while in others the increase in the effective local concentration of protein may account for the effects observed. The ability of DNA to form loops is affected by the distance between binding sites; by the DNA sequence, which determines deformability and bendability; and by the presence of other proteins that exert an influence on the conformation of a particular sequence. Alteration of the stability of DNA loops and/or protein-DNA binding by extra- or intracellular signals provides responsivity to changing metabolic or environmental conditions. The fundamental property of site-specific protein binding to DNA can be combined with protein-protein and protein-ligand interaction to generate a broad range of physiological states.

DNA↗
PubMed1991

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.

Arginine↗
PubMed1991

Characterization of mutations in oligomerization domain of Lac repressor protein.

A series of mutant lac repressor proteins at positions 281 or 282 was isolated for detailed characterization. Although Cys281 modification by sulfhydryl reagents abrogates pH effects on inducer binding and diminishes operator binding (Daly, T. J., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 5468-5474), substitution at this site by alanine, serine, phenylalanine, isoleucine, or methionine did not abolish completely the pH shift nor affect operator affinity. Thus, ionization of the sulfhydryl residue does not account fully for the alterations in inducer affinity and cooperativity of binding observed with elevated pH. Substitution for Cys281 did, however, alter the kinetic parameters for inducer association with the protein. The polarity of the side chain at 281 influenced the rates of sugar binding, presumably by altering the rate of opening/closing of the binding site. Furthermore, the presence of the branched side chain of isoleucine at position 281 disrupted oligomerization of the repressor. In contrast to the tolerance for substitution at 281, the only amino acid side chain exchanges for Tyr282 which yielded tetrameric protein with near normal operator binding characteristics were phenylalanine and leucine; this result is consistent with studies of suppressed nonsense mutations at position 282 which indicated repression occurred only for the corresponding substitutions (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 221, 295-318). Despite the tetrameric character of the Y282F mutant protein, the pH dependence and cooperativity of inducer binding for this mutant protein were altered. All amino acid substitutions other than phenylalanine and leucine at this position resulted in either monomeric protein or no detectable repressor in the cell. Thus, the hydrophobic character of the side chain at position 282 is essential for tetramer formation, and the phenyl ring alone alters inducer binding parameters. The monomeric mutant proteins with substitutions for Tyr282 exhibited lower stability than their tetrameric counterparts, and the absence of dimer formation suggests alterations at this site affect both dimer and tetramer interfaces. Based on previous genetic studies and our detailed mutant characterization, the region encompassing 281 and 282, indicated by secondary structure prediction to be a turn or coil, is essential for oligomer formation and additionally exerts a strong influence on the dynamic properties of the protein, presumably mediated by interactions at the subunit interface which regulate the rate of opening and closing of the inducer binding cleft.

Amino Acid Sequence↗
PubMed1991

Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation.

Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs (Ogata, R., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4973-4976). The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.

Autoradiography↗
PubMed1991

Identification of amino acids in lac repressor protein cross-linked to operator DNA specifically substituted with bromodeoxyuridine.

Amino acids in lac repressor protein which form cross-links to lac operator DNA specifically substituted with bromodeoxyuridine (BrdU) have been identified. Five sites of cross-linking in BrdU-substituted operator DNA were found at positions +3, +4, +14, +18, and +19 relative to the initiation site for transcription (Wick, K.L., and Matthews, K.S. (1991) J. Biol. Chem. 266, 6106-6112). Irradiation of complexes of repressor and each of these five singly substituted operator DNAs was executed under large scale conditions to generate sufficient complex for proteolysis, separation of the peptide-DNA, and peptide sequencing. The DNAs substituted with BrdU for thymidine at positions +3, +18, and +19 yielded cross-links to the peptide spanning residues 23-33, with the cross-link identified at His-29. Substitution at position +14 resulted in a cross-link to Tyr-17 within the peptide containing amino acids 13-22. These results are consistent with the structure determined by NMR and molecular dynamics calculations of the NH2-terminal headpiece-symmetric operator complex (Lamerichs, R.M.J.N., Boelens, R., van der Marel, G.A., van Boom, J.H., Kaptein, R., Buck, F., Fera, B., and Rüterjans, H. (1989) Biochemistry 28, 2895-2991; de Vlieg, J., Berendsen, H.J.C., and van Gunsteren, W.F. (1989) Proteins 6, 104-127). This structure indicates proximity of His-29 in the major groove to thymidines at positions +3 and +4. Since base pairs at positions +18 and +19 occupy symmetrical positions to +3 and +4 in the promoter distal region of the operator, it would be anticipated that cross-links similar to the +3 and +4 positions would form at these sites; this prediction is not borne out by the behavior at +4/+18, as no peptide could be identified cross-linked to DNA substituted at +4. Molecular dynamics simulations and the NMR data indicate that Tyr-17 interacts with the thymine at position +8, which is symmetrically related to position +14. Although BrdU-associated strand scission at +8 is protected, this site does not cross-link with bound lac repressor; inversely, DNA substituted with BrdU at +14 cross-links to repressor, but is not protected from strand scission by the presence of the protein. These differences at symmetrically related nucleotide positions (+4 versus +18, +8 versus +14) reflect the inherent asymmetry in the interaction. The identification of amino acids in proximity to specifically substituted sites confirms that positions of several amino acids in the intact protein-operator complex correspond to those in the structure of the NH2-terminal headpiece-operator DNA complex.

Amino Acid Sequence↗
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↗
PubMed1991

Evidence for leucine zipper motif in lactose repressor protein.

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.

Amino Acid Sequence↗
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

Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy.

High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.

Dansyl Compounds↗
PubMed1990

Effect of amino acid alterations in the tryptophan-binding site of the trp repressor.

Mutations in the tryptophan-binding site of the trp repressor have been generated using site-directed mutagenesis. The selection of sites for alteration was based on the three-dimensional x-ray crystallographic structure (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L., and Sigler, P. B. (1985) Nature 317, 782-786). The changes generated include Thr-44 to Ala (T44A), Arg-54 to Leu (R54L), Arg-54 to Lys (R54K), Arg-84 to Leu (R84L), and Arg-84 to Lys (R84K). The mutant proteins were purified and characterized in detail for their binding properties. Both tryptophan and operator DNA affinities for all five mutants were decreased. The R84L, R54K, and R54L mutants exhibited increases in Kd for operator DNA relative to wild-type repressor ranging from approximately 10(3) to approximately 10(4), while R84K and T44A exhibited increases of 10- to 100-fold. This diminution in DNA binding activity derives at least in part from diminished affinity for tryptophan, although decreased affinity for nonspecific DNA was also observed for these mutant proteins. Tryptophan binding was not detectable by equilibrium dialysis for most of the mutant proteins, but this activity was measurable for several of the altered proteins by monitoring the fluorescence decrease associated with the displacement of 1-anilino-8-naphthalenesulfonate from the tryptophan-binding site (Chou, W.-Y., and Matthews, K. S. (1989) J. Biol. Chem. 264, 18314-18319). These measurements revealed that tryptophan bound to R84K, T44A, and R84L repressors with Kd values 1.5- to 13-fold higher than that for wild-type repressor. It was not possible to detect tryptophan binding to R54K and R54L even using the fluorescence assay. Circular dichroism spectra demonstrated that the mutants and the wild-type repressor possess similar secondary structural features. The results of this selected substitution in the tryptophan-binding site are readily interpreted based on the x-ray structural analysis.

Amino Acid Sequence↗
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↗
PubMed1989

Serine to cysteine mutations in trp repressor protein alter tryptophan and operator binding.

The tryptophan repressor regulates expression of the aroH, trpEDCBA, and trpR operons in Escherichia coli. The protein contains no cysteine residues, and the presence of this reactive side chain would allow introduction of spectral probes to monitor binding reactions. Three mutant trp aporepressors, each with a point mutation from serine to cysteine, were produced at positions 67, 86, and 88 by oligonucleotide-directed site-specific mutagenesis. This single conservative substitution affected both tryptophan and operator DNA affinities in all three purified proteins. Cysteine substitution for serine at position 67 decreased tryptophan binding by approximately 6-fold and the operator DNA affinity by approximately 50-fold. The proximity of this amino acid to Gln-68 which is involved in binding to operator DNA (Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Nature 335, 321-329) may account for this effect. Substitution at position 86 diminished tryptophan binding by approximately 4-fold and operator DNA binding by approximately 130-fold. The participation of Ser-86 in the hydrogen bond network required for operator binding (Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Nature 335, 321-329) presumably accounts for the DNA binding effects. The diminished corepressor activity in these two mutants may derive from distortions of the binding region, as the tryptophan and DNA binding sites are intimately related. The mutation at position 88 altered tryptophan binding the most of the three mutants (approximately 18-fold) and operator binding least (approximately 12-fold). Ser-88 forms a hydrogen bond with the amino group of bound tryptophan (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L., and Sigler, P. B. (1985) Nature 317, 782-786), and alteration of the geometry of the side chain would be anticipated to perturb the topology of the binding site. The diminished operator affinity may derive from improper alignment of the tryptophan ligand, crucial for high affinity operator binding (Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Nature 335, 321-329).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗