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K S Matthews

Publications and source records attributed to K S Matthews.

At least 19 recordsLinked to original sources
PubMed2001

Protein-DNA binding correlates with structural thermostability for the full-length human p53 protein.

Full-length p53 protein purified from Escherichia coli in the unmodified, "latent" form was examined by several methods to correlate thermal stability of structure with functional DNA binding. Structure prediction algorithms indicate that the majority of beta-sheet structure occurs in the p53 core DNA binding domain. Circular dichroism spectra demonstrate that the intact protein is surprisingly stable with a midpoint for the irreversible unfolding transition at approximately 73 degrees C. Significant beta-sheet structural signal remains even to 100 degrees C. The persistent beta-sheet CD signal correlates with significant DNA binding (K(d) approximately nM range) to temperatures as high as 50 degrees C. These data confirm the ability of the DNA binding domain in the full-length "latent" protein to bind consensus dsDNA targets effectively in the absence of activators over a broad temperature range. In addition, we demonstrate that Ab1620 reactivity is not directly correlated with the functional activity of the full-length protein since loss of this epitope occurs at temperatures at which significant specific DNA binding can still be measured.

Antibodies, Monoclonal↗
PubMed2000

Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein.

The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail the interplay between DNA and protein sequence, we have examined binding of variant lac operator DNA sequences to a series of mutant lactose repressor proteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains. Variant operators differed from the wild-type operator, O(1), in spacing and/or symmetry of the half-sites that contact the LacI N-terminal DNA binding domain. Binding of wild-type and mutant proteins was affected differentially by variations in operator sequence and symmetry. While the mutant series exhibits a 10(4)-fold range in binding affinity for O(1) operator, only a approximately 20-fold difference in affinity is observed for a completely symmetric operator, O(sym), used widely in studies of the LacI protein. Further, DNA sequence influenced allosteric response for these proteins. Binding of this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pair sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence may establish the binding environment for protein and determine both affinity and potential for allostery.

Bacterial Proteins↗
PubMed2000

Generation of an AraC-araBAD promoter-regulated T7 expression system.

An alternative and facile delivery system for T7 RNA polymerase has been devised and constructed. T7 gene 1 has been placed under control of the araBAD promoter element regulated by the AraC protein. Cotransformation of the resultant plasmid, pTara, with one containing a target gene under T7 promoter-regulated expression potentially allows repression by glucose and induction by arabinose in the range of 0.5 to 20 mM sugar concentration. To demonstrate the efficacy of this expression system, the p53 gene under T7 promoter control in two different plasmids was expressed in Escherichia coli using pTara as the source of T7 RNA polymerase. Repression and induction of p53 were achieved in both a lower and higher copy number plasmid, although the levels of induction were higher with the lower copy number expression vector. Cotransformation of an expression plasmid with pTara provides a low-cost method of T7 RNA polymerase-regulated expression that can be fine-tuned using glucose and arabinose concentrations to balance protein expression with potential solubility or toxicity problems.

Arabinose↗
PubMed1999

Glycine insertion in the hinge region of lactose repressor protein alters DNA binding.

Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose repressor protein (LacI). Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly. All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines. Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence.

Amino Acid Sequence↗
PubMed1999

Ligand-induced conformational changes in lactose repressor: a phosphorescence and ODMR study of single-tryptophan mutants.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations.

Amino Acid Substitution↗
PubMed1999

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein.

Bacterial Proteins↗
PubMed1999

Substitutions at histidine 74 and aspartate 278 alter ligand binding and allostery in lactose repressor protein.

In the inducer-bound structure of the lac repressor protein, the side chains of H74 and D278 are positioned to form an ion pair between monomers that appears to be disrupted upon operator binding (Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). A series of single substitutions at H74 and D278 and a double mutant, H74D-D278H, were generated to determine the influence of this interaction on ligand binding and allostery in lac repressor. Introduction of apolar amino acids at H74 resulted in distinct effects on ligand binding. Alanine and leucine substitutions decreased operator binding, while tryptophan and phenylalanine increased affinity for operator DNA. Introduction of a negatively charged residue at position 74 in H74D had minimal effects, and "inverting" the side chains in H74D/D278H did not significantly alter inducer or operator binding at neutral pH. In contrast, all substitutions of D278 increased affinity for operator DNA and diminished inducer binding. These observations can be interpreted in the context of the Monod-Wyman-Changeux model. If a salt bridge were essential for stabilizing or destabilizing the inducer-bound conformation, a mutation at either residue that interrupts this interaction should have a similar effect on allostery. Because the type and degree of alteration in ligand binding properties depended on the nature of the substitution at these residues, the individual roles played by H74 and D278 in lac repressor allostery appear more important than their direct contact across the monomer-monomer interface.

Allosteric Regulation↗
PubMed1998

Kinetic and thermodynamic studies of purine repressor binding to corepressor and operator DNA.

The kinetic and thermodynamic parameters for purine repressor (PurR)-operator and PurR-guanine binding were determined using fluorescence spectroscopy and nitrocellulose filter binding. Operator binding affinity was increased by the presence of guanine as demonstrated previously (Choi, K. Y., Lu, F., and Zalkin, H. (1994) J. Biol. Chem. 269, 24066-24072; Rolfes, R. J., and Zalkin, H. (1990) J. Bacteriol. 172, 5637-5642), and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by approximately 10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is approximately 3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences.

Bacterial Proteins↗
PubMed1998

Comparison of simulated and experimentally determined dynamics for a variant of the Lacl DNA-binding domain, Nlac-P.

Recent advances in the experimentally determined structures and dynamics of the domains within LacI provide a rare context for evaluating dynamics calculations. A 1500-ps trajectory was simulated for a variant of the LacI DNA-binding domain, which consists of the first three helices in LacI and the hinge helix of the homologous PurR. Order parameters derived from dynamics simulations are compared to those obtained for the LacI DNA-binding domain with 15N relaxation NMR spectroscopy (Slijper et al., 1997. Biochemistry. 36:249-254). The MD simulations suggest that the unstructured loop between helices II and III does not exist in a discrete state under the conditions of no salt and neutral pH, but occupies a continuum of states between the DNA-bound and free structures. Simulations also indicate that the unstructured region between helix III and the hinge helix is very mobile, rendering motions of the hinge helix essentially independent of the rest of the protein. Finally, the alpha-helical hydrogen bonds in the hinge helix are broken after 1250 ps, perhaps as a prelude to helix unfolding.

Amino Acid Sequence↗
PubMed1998

Lactose repressor protein: functional properties and structure.

The lactose repressor protein (LacI), the prototype for genetic regulatory proteins, controls expression of lactose metabolic genes by binding to its cognate operator sequences in E. coli DNA. Inducer binding elicits a conformational change that diminishes affinity for operator sequences with no effect on nonspecific binding. The release of operator is followed by synthesis of mRNA encoding the enzymes for lactose utilization. Genetic, chemical and physical studies provided detailed insight into the function of this protein prior to the recent completion of X-ray crystallographic structures. The structural information can now be correlated with the phenotypic data for numerous mutants. These structures also provide the opportunity for physical and chemical studies on mutants designed to examine various aspects of lac repressor structure and function. In addition to providing insight into protein structure-function correlations, LacI has been utilized in a wide variety of applications both in prokaryotic gene expression and in eukaryotic gene regulation and studies of mutagenesis.

Amino Acid Sequence↗
PubMed1997

Ligand-induced conformational changes in lactose repressor: a fluorescence study of single tryptophan mutants.

A key element in the ability of lac repressor protein to control transcription reversibly is the capacity to assume different conformations in response to ligand binding. To investigate regions of the protein involved in these conformational changes, mutant repressor proteins containing single tryptophans were created by mutating each of the two native tryptophan residues to tyrosine and changing the residue of interest to tryptophan. Tryptophans substituted in the following locations were highly accessible to quenchers with no changes in fluorescence or quenching properties in the presence of ligands: in the N-terminal helix-turn-helix for Y7, at the junction between the N-terminus and N-subdomain for L62, in the N-subdomain of the monomer-monomer interface for residue E100 or Q117, or at the C-terminal region for K325. Tryptophan at position F226 in the C-subdomain subunit interface was only moderately exposed to quenchers and unresponsive to ligands. In contrast, the fluorescence and quenching properties of single tryptophans placed in the central region of the protein were affected by ligands. Inducer binding altered the accessibility to quencher for tryptophan at H74 or F293, but no changes were detected upon binding operator. Exposure of tryptophan at the position occupied by Y273 was affected by both inducer and operator, indicating alterations in this region by both ligands. These results suggest that, in the areas of the lac repressor probed by these substitutions, the inducer-bound form differs from the conformation of the unliganded form.

Bacterial Proteins↗
PubMed1997

Designed disulfide between N-terminal domains of lactose repressor disrupts allosteric linkage.

Substitution of Cys for Val at position 52 of the lac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site. This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lac repressor-operator complex (Schumacher, M. A., Choi, K. Y., Zalkin, H., and Brennan, R. G. (1994) Science 266, 763-770; Lewis, M., Chang, G., Horton, N.C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Science 271, 1247-1254). The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized. In the reduced form, V52C bound operator DNA with slightly increased affinity. Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with approximately 6-fold higher affinity than wild-type protein. Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor. In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding. In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mM. Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding.

Allosteric Regulation↗
PubMed1997

Combinatorial mutations of lac repressor. Stability of monomer-monomer interface is increased by apolar substitution at position 84.

To examine the monomer-monomer subunit interface in the lac repressor, a mutation that generates dimeric protein (deletion of C-terminal amino acids to disrupt the dimer-dimer interface) has been combined with amino acid substitutions that alter the monomer-monomer interface (substitution at Lys84 or Tyr282). Dimeric proteins with significantly increased stability to urea denaturation were formed by the introduction of the apolar amino acids Ala or Leu in lieu of Lys84 in concert with the deletion of 11 C-terminal amino acids. K84A/-11 deletion protein retained wild-type affinity for operator DNA, while K84L/-11 deletion protein displayed operator affinity similar to its parent tetramer. To assess further the influence of monomer-monomer interface stability on assembly and DNA binding, triple mutants were generated with Y282D, an alteration that disrupts assembly completely in the wild-type background. The triple mutants were dimeric, but they exhibited diminished dimer stability to urea denaturation and decreased operator affinity compared with the double mutations. These results demonstrate directly the stabilizing influence of apolar substitution at position 84 on the monomer-monomer interface.

Bacterial Proteins↗
PubMed1997

Differences in water release with DNA binding by ultrabithorax and deformed homeodomains.

The amino acid sequences of the homeodomains (HD) within the Ultrabithorax (Ubx) and Deformed (Dfd) proteins from Drosophila melanogaster are highly conserved despite distinct genetic regulatory functions for these proteins in embryonic development. We reported recently that Ubx-HD binding to a single target site displayed significantly increased affinity and greater salt concentration dependence at lower pH; in contrast, Dfd-HD did not show pH dependence in its DNA binding properties [Li, L., et al. (1996) Biochemistry 35, 9832-9839]. We demonstrate in this study that water activity differentially affects Ubx-HD and Dfd-HD DNA binding affinity. The sensitivity of the protein-DNA binding constant to osmotic pressures generated by neutral solutes was measured, and the formation of the Ubx-HD-DNA complex is associated with significantly greater water release than that of the Dfd-HD-DNA complex. No influence of pH on water release was detected for either HD. Experiments with chimeric Ubx-Dfd homeodomains demonstrated that the C-terminal region of the Ubx-HD is the primary determinant for the greater water release associated with DNA binding for this protein. DNA sequences do not exert a significant effect on the magnitude of water release associated with protein-DNA binding for Ubx-HD and the chimeric HD, UDU.

Amino Acid Sequence↗
PubMed1996

pH-dependent enhancement of DNA binding by the ultrabithorax homeodomain.

Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. We report in the present study that Ubx-HD binding to a single optimal target site displayed significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA was unaffected by pH. Results from studies of chimeric Ubx-Dfd homeodomains showed that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH was greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo.

Amino Acid Sequence↗
PubMed1995

Protective effect of vitamin E on lymphocyte growth capacity during incubation in vitro.

Vitamin E, an essential lipid-soluble micronutrient, plays an important role in the immune system and serves as an immunostimulant in geriatric subjects. Using an in vitro incubation to simulate aging processes, we find that vitamin E mitigates loss of growth capacity in lymphocytes. Vitamin E presence during in vitro incubation did not affect significantly the level of peroxidation, the effects of exogenous IL-2, PGE2, or indomethacin, nor levels of IL-2 production. Thus, the preservative effect on lymphocyte growth by vitamin E is not due primarily to its antioxidant function or to interleukin-2 or prostaglandin effects. The decreased growth capacity generated by in vitro incubation is accompanied by a variety of cellular alterations, including decreased CD5 surface antigen, enhanced suppression by adherent cells, and impaired communication between lymphocytes and adherent cells. The decrease in CD5 surface antigen correlates inversely with the cell density required for maximal cell proliferation, and the diminished CD5 levels were unaltered by vitamin E presence during the aging process. In contrast, protection of T-cell proliferative capacity by vitamin E in vitro correlates with diminished suppression by adherent cells and normalized interaction between lymphocytes and adherent cells.

Adult↗