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K S Matthews

Publications and source records attributed to K S Matthews.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1995

Human lymphocytes incubated in vitro share multiple characteristics with geriatric-derived lymphocytes: a potential in vitro model for aging?

Aging involves a complicated set of parallel reactions that result in multiple cellular and organismic changes and may eventuate in chronic illness. In the immune system, several alterations that correlate with age have been established. In the present study, we report the results of incubating lymphocytes in vitro in whole blood and, employing measures known to be age-dependent, compare these cells 'aged' in vitro with cells from geriatric patients aged in vivo. Cells from blood aged in vivo and incubated in vitro share a number of common characteristics that include decreased growth capacity, shifted growth patterns, increased suppression by adherent cells, decrease in CD5 surface antigen, similar responses to addition of exogenous IL-1, IL-2, PGE2, or indomethacin, and similar production of PGE2. Differences found between in vivo aging and in vitro incubation are IL-2 plasma levels and IL-2 production by activated cells monitored in conditioned minimal medium. Based on these observations, this in vitro system provides a simple method to generate cells that exhibit a very significant subset, but not all, of the characteristics associated with in vivo aging in lymphocytes.

Adult↗
PubMed1995

Role of Asp274 in lac repressor: diminished sugar binding and altered conformational effects in mutants.

The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site. Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M). Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions. The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins. Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor. From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding.

Aspartic Acid↗
PubMed1995

Characterization of mutants affecting the KRK sequence in the carboxyl-terminal domain of lac repressor.

The lac repressor carboxyl-terminal region is required for tetramer assembly and protein stability. To further investigate this region, especially the unusual sequence KRK, four deletion mutants eliminating the carboxyl-terminal 34, 35, 36, and 39 amino acids and five substitution mutants at the position of Arg-326, R326K, R326A, R326E, R326L, and R326W, were constructed using site-specific mutagenesis. The -34-amino-acid (aa) mutant, missing the most carboxyl-proximal lysine from the KRK sequence, exhibited lower affinity for both operator and inducer and lower protein stability than dimeric proteins studied previously. The -35-aa mutant with RK missing, as well as -36 aa and -39 aa, for which the entire KRK sequence was deleted, yielded inactive polypeptides that could be detected only by monoclonal antibody for lac repressor. In the Arg-326 mutant proteins, operator binding affinity was decreased by approximately 6-fold, the shift in inducer binding at elevated pH was diminished, and protein stability was decreased. Dramatic decreases in protein expression and stability occurred with substitution at position 326 by glutamate, leucine, or tryptophan. These results suggest that Arg-326 plays an important role in the formation of the proper tertiary structure necessary for inducer and operator affinity and for protein stability.

Amino Acid Sequence↗
PubMed1994

Glucose and insulin responses in isolated human lymphocytes reflect in vivo status: effects of VLCD treatment.

Human lymphocyte growth response to mitogen was examined in vitro under different conditions to monitor aspects of carbohydrate utilization in three groups: obese nondiabetic, obese/NIDDM, and normal-weight individuals. Although lymphocyte growth capacity in minimal medium for these three groups was found to be similar at a glucose concentration of 72 micrograms/ml in the absence of insulin or glycerol, differences in mitogen-stimulated growth were observed at lower glucose concentrations or in the presence of insulin or glycerol. Interestingly, these metabolic alterations in lymphocytes can be normalized by treating obese and obese/NIDDM subjects with a very low calorie diet (VLCD) regimen. The results of this study indicate that lymphocyte culture in a defined medium may provide a mechanism to examine different metabolic states and to evaluate treatment regimens (diet, exercise, etc.) for obese and NIDDM subjects.

Blood Glucose↗
PubMed1994

Subunit dissociation affects DNA binding in a dimeric lac repressor produced by C-terminal deletion.

The reduction in apparent operator binding affinity found for dimeric lac repressor proteins produced by disruption of the C-terminal coiled-coil interaction has been proposed to derive from thermodynamic linkage between dimer-monomer and protein-DNA equilibria [Brenowitz et al. (1991) J. Biol. Chem. 266, 1281]. To explore this linkage, we have employed two dimeric proteins, a deletion mutant (-11 aa) missing 11 amino acids at the C-terminus that has diminished apparent operator binding affinity [Chen & Matthews (1992) J. Biol. Chem. 267, 13843] and a mutant (R3) that binds to operator with wild-type affinity in which the C-terminal leucine heptad repeats of lac repressor were replaced by the GCN4 dimerization sequence [Alberti et al. (1993) EMBO J. 12, 3227; Chen et al. (1994) J. Biol. Chem. (in press)]. To avoid the complexities of working at the low concentrations of protein required by the high affinity between the monomer subunits, urea denaturation studies were undertaken to determine the free energy change(s) for dissociation and/or unfolding. Under denaturing conditions, dimer dissociation and monomer unfolding were found to be concerted processes, and the free energy change for the overall process of dimer to unfolded monomer was derived from these experiments for the two dimeric proteins. A monomeric mutant (Y282D) of the lactose repressor was examined to determine the free energy change of protomer unfolding. From the combination of these data, the Kd for -11 aa dimer dissociation was determined to be 7.7 x 10(-8) M, and the corresponding value for R3 protein was 3.2 x 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1994

Wild-type operator binding and altered cooperativity for inducer binding of lac repressor dimer mutant R3.

Substitution of the C-terminal leucine heptad repeat region of the normally tetrameric lactose repressor by the leucine heptad repeat dimerization domain of GCN4 protein resulted in cell extracts containing protein, designated R3, which behaved as a dimer based on gel retardation analysis of DNA binding (Alberti, S., Oehler, S., von Wilcken-Bergmann, B., and Müller-Hill, B. (1993) EMBO J. 12, 3227-3236). We have purified this R3 protein and characterized its properties in comparison with the wild-type repressor. R3 protein elutes from a molecular sieve with a Stokes radius characteristic of a dimer and a deduced molecular mass of 66 kDa. Unlike other dimeric repressors, produced by deletion or mutation in the leucine heptad repeat region, which display reduced apparent operator affinity, R3 binds to operator DNA sequences with wild-type equilibrium and kinetic properties. Although inducer affinity at neutral pH is similar for R3 and wild-type protein, at elevated pH the R3 protein undergoes a slightly smaller decrease in affinity and exhibits minimal cooperativity in sugar binding compared with the wild-type protein. Interestingly, in the presence of operator DNA, a state in which inducer binding to wild-type repressor is also of reduced affinity and slightly cooperative, R3 binding affinity is decreased to a greater extent, and the protein displays higher cooperativity than wild-type repressor. Consistent with inducer binding data in the presence of operator, the release of operator from R3 protein requires a higher sugar concentration than wild-type protein. These results are interpreted in the context of alterations involving the subunit interface which affect the allosteric behavior of the repressor protein.

Allosteric Regulation↗
PubMed1994

Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor.

To explore the roles of three aspartate residues, Asp88, Asp130, and Asp274, found in the proposed inducer binding site of lac repressor [Sams, C. F., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1984) Nature 310, 429-430], each site was substituted with alanine, glutamate, lysine, or asparagine by site-specific mutagenesis. The mutations at the Asp88 site resulted in a 5-13-fold decrease in inducer binding affinity, largely due to an increase in the inducer dissociation rate constants for these mutants. In addition, the mutant proteins Asp88-->Ala and Asp88-->Lys exhibited altered allosteric behavior for inducer binding. These data conflict with the original hypothesis placing Asp88 in the inducer binding site, but are in agreement with a recent model that places this amino acid close to the subunit interface involved in cooperativity associated with inducer binding [Nichols, J. C., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. Substitution at Asp130 did not alter the inducer binding affinity nor other binding activities. Thus, this amino acid is not crucial in the binding to beta-substituted monosaccharides or in protein function. In stark contrast, all mutant proteins with substitutions at the Asp274 site exhibited no detectable inducer binding. With the exception of Asp274-->Lys, the structures of these mutant proteins appear to be similar to wild-type. The data demonstrate that Asp274 plays a crucial role in inducer binding of this transcriptional regulator.

Allosteric Regulation↗
PubMed1994

Construction of a dimeric repressor: dissection of subunit interfaces in Lac repressor.

Formation of the lactose repressor tetramer is postulated to involve two subunit interfaces, one primarily contributing to monomer-monomer assembly to dimer and the second to dimer-dimer association to tetramer. The latter interface requires a heptad repeat of three leucines at the C-terminus of lac repressor that is presumed to form an abbreviated coiled-coil motif [Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., & Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Krämer, H., & Müller-Hill, B. (1991) New Biol. 3, 57-62; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. To strengthen the dimer-dimer interface, this motif was extended by the addition of one and two leucine heptad repeat units to the C-terminus by site-specific insertion mutagenesis. The tetrameric products displayed operator and inducer affinity essentially indistinguishable from the wild-type repressor. In order to probe the effect of the elongated coiled-coil on assembly of the repressor tetramer, the other of the two postulated subunit interfaces was disrupted by introducing a point mutation (Y282D) that yields a monomeric protein in the wild-type background. Both elongated mutant repressors were able to assemble into dimeric species, apparently due to the strengthened subunit association at the C-terminal region compared to the wild-type repressor. These results further confirm the role of a coiled-coil structure in the formation of tetramer in the lac repressor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Dietary deprivation of B-vitamins reflected in murine splenocyte proliferation in vitro.

The effect of murine dietary deprivation of specific B-vitamins on in vitro splenocyte proliferative response in short-term tissue culture was examined in serum-free, protein-free, minimal medium. Using different media formulations, incorporation of [3H]thymidine was used to monitor the relative growth capacity of phytohemagglutinin-stimulated splenocytes from mice exposed to different diets. The results demonstrate that the growth deficit observed in vitro in the absence of specific vitamin(s) in the medium correlates only to those specific vitamin(s) eliminated from the diet. Dietary repletion is followed by restoration of a normalized response in vitro. The finding that murine dietary experience is reflected in vitro in splenocyte proliferative response monitored by [3H]thymidine incorporation suggests that human dietary experience might be monitored by similar analysis of human peripheral blood lymphocyte response.

Animals↗
PubMed1994

trp repressor mutations alter DNA complex stoichiometry.

We have examined the interaction of a series of mutant trp repressors with various operator DNA sequences using gel retardation. Binding to 40 base pairs (bp) TrpEDCBA operator yielded patterns distinct from the wild-type protein for superrepressors EK13, EK18, and EK49, with a protein-DNA complex of higher stoichiometry (three dimers/operator) than observed for wild-type repressor (two dimers/operator). This higher stoichiometry complex may contribute to the enhanced binding affinity and higher protein-operator stability observed for the superrepressors. In contrast, DN46 displayed the same complexes characteristic of the wild-type protein, although the complex of a single dimer with operator was more prominent in the DN46 binding pattern than wild-type despite higher apparent affinity of this protein for TrpEDCBA operator than wild-type protein. The binding of AV77 was indistinguishable from the wild-type protein. Similar patterns to that found for TrpEDCBA were also observed for the 40-bp aroH operator and symmetrized derivatives of TrpEDCBA for these superrepressors. Binding of EK13, EK18, and EK49 superrepressors to half-site DNAs, composed of 20 bp of TrpEDCBA sequence coupled with 20 bp of lac operator sequence, yielded 2:1 complex as the primary product with no detectable 3:1 complex; thus, two half-sites appear to be required for generation of the 3:1 complex. Mutation in the tryptophan-binding site can also generate higher order complexes with TrpEDCBA DNA as demonstrated by the binding of VA58; the presence of 3:1 complex with this protein was also dependent on the presence of two half-sites. In addition to effects of sequence changes in the protein, the ligand employed can influence the binding pattern, as demonstrated for EK49 and VA58 using 5-methyl-tryptophan; the 3:1 complex is produced more prominently and at lower protein concentration for both mutants. It is apparent from these data that binding of the trp repressor to DNA is influenced by the operator sequence, the nature of the corepressor, as well as interactions (perhaps involving the N-terminal regions) that occur within and between the dimeric structure of this protein.

Bacterial Proteins↗
PubMed1993

Characterization of the two tryptophan residues of the lactose repressor from Escherichia coli by phosphorescence and optical detection of magnetic resonance.

The native lactose repressor from Escherichia coli (Lac Rep) and two single-point mutants, W220Y and W201Y, were investigated using low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Emission from two tryptophan residues was evident in the phosphorescence spectrum of native Lac Rep at 77 K. Using the single-point mutants, the triplet-state properties of tryptophans 201 and 220 were obtained independently. Trp 220 was characterized as a partially solvent-exposed residue (0,0 band centered at 409.5 nm), while tryptophan 201 exhibited the properties of a buried residue (0,0 band centered at 413.5 nm). Both single-point mutant proteins experienced changes in tryptophan triplet-state properties as a result of binding either of two inducer sugars: isopropyl beta-D-thiogalactoside, a monosaccharide, or melibiose, a disaccharide. Putative singlet-singlet energy transfer from tryptophan 220 to tryptophan 201 was also investigated, but the quantitative results must be viewed with some caution.

Energy Transfer↗
PubMed1993

Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size.

A series of chemically synthesized trp and mutant operator DNAs was employed to examine trp repressor binding. Although only a single repressor-operator complex was observed for most DNAs as reported previously, varying DNA sequence revealed two retarded complexes with an additional band of faster mobility. The relative intensity of the two retarded bands with varying repressor concentrations suggests that cooperative interactions between dimers may occur in the formation of the predominant repressor-operator complex. Direct stoichiometry measurements demonstrated that a 2:1 stoichiometry (two dimers per operator) is found in the primary repressor-operator complex band and that a 1:1 stoichiometry is observed, when present, for the minor repressor-operator complex band of faster mobility. Similar retardation patterns with a single complex of 2:1 stoichiometry were observed for 40-base pair (bp) trp operators corresponding to TrpEDCBA, aroH, and Trp-PL (a derivative of TrpEDCBA with increased symmetry) operator sequences as well as to hybrid operators containing a half-binding site from TrpEDCBA in conjunction with lac operator sequences, although the apparent affinity for the half-site DNAs was diminished by 10-fold. In contrast, the prominence of the 1:1 dimer-operator complex for Trp-PR, a different derivative of TrpEDCBA with increased symmetry, suggests that sequence context may diminish cooperativity between dimers. The stoichiometry observed was also dependent on the length of TrpEDCBA operator DNA used, shifting from primarily 2:1 for 40- and 33-bp TrpEDCBA DNA to primarily 1:1 for 29-, 26-, and 20-bp TrpEDCBA DNAs. In addition, the stability of the repressor-operator complex to electrophoresis is reduced for DNA lengths of 33, 29, and 26 bp. Based on the binding data and footprinting patterns for two hybrid 40-bp TrpEDCBA/lac half-binding site DNAs, it appears that repressor associates tightly to the specific trp half-site, whereas the nonspecific half of the DNA is more loosely bound. These results suggest that repressor dimer-dimer interaction may be an important feature in the trp repressor-operator interaction.

Allosteric Regulation↗
PubMed1993

Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a sequence-dependent manner.

In order to explore DNA sites influenced by the trp repressor-operator interaction, bromodeoxyuridine (BrdU) was chemically incorporated into the TrpEDCBA, TrpR, and aroH operators at selected thymidine positions. Different patterns of repressor protection from strand scission in the two halves of complexes with the TrpEDCBA, TrpR, and aroH operators suggest different local environments despite the highly symmetric sequences. Although protection was observed at multiple sites in the operators in the presence of repressor, UV irradiation did not lead to a cross-linked repressor-operator complex. This result indicates the absence of close contacts in the major groove between suitable repressor residues and the 5-methyl of thymidines. Upon trp repressor binding and UV irradiation, in addition to protection from strand scission, multiplets were observed at some sites, notably within CTAG sequences in the BrdU-substituted operators. This phenomenon (termed band migration) may result from distortion by the trp repressor of the BrdU-substituted operator DNA and consequent exposure of different sites along the backbone to strand scission. Interestingly, UV footprinting of two BrdU-substituted TrpEDCBA variant operators showed different patterns when base pair symmetry was matched to each side of the symmetry axis. These observations suggest that alterations in the UV photolysis pattern in response to protein binding result from DNA structural alterations that are sequence dependent.

Bacterial Proteins↗