R-3 Repository :: Browsing by Author "Matthews, Kathleen S."

Browsing by Author "Matthews, Kathleen S."

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A simple and effective model to study insulin resistance in obesity and diabetes and the processes and mechanisms of in vitro aging using lymphocyte culture
(1994) Tu, Keyin; Matthews, Kathleen S.
Cell culture provides a simple and effective system to study metabolic and cellular processes required for cellular growth in vitro. In this study, we demonstrate that lymphocyte culture can be used as a model system to study the mechanism of insulin resistance (obesity/diabetes) as well as the process of aging of cells from the immune system. Our results indicate that metabolic differences can be observed in lymphocytes from obese subjects and that different degrees of alteration in response can be detected between obese nondiabetic and diabetic subjects. Furthermore different effects of insulin resistance can be reflected faithfully in lymphocyte biochemistry in culture. In isolated lymphocytes from normal weight subjects, G3PDH (an enzyme at the intersection of glycolytic and lipogenic pathways) activity increases in the presence of insulin. Augmented G3PDH activity requires new protein synthesis and involves the inositol triphosphate-diacylglycerol signalling system. However, in obese subjects for whom insulin resistance in vitro can be demonstrated, the extent of insulin stimulation of G3PDH activity is decreased compared to normal weight individuals. These results suggest that G3PDH activity is dependent upon the metabolic state of the subjects from which the cells are obtained. Dietary restriction for obese subjects normalizes insulin and glycerol responses and insulin effects on G3PDH activity. These results demonstrate that the lymphocyte can serve in vitro as a model to reflect organismal metabolism in obesity/diabetes. Alterations of lymphocytes due to aging contribute significantly to changes in immune function. In present study, an artificial aging environment was introduced using whole blood. In this environment, we demonstrate that lymphocytes undergo degeneration and aging processes similar to those observed in vivo. Similar changes in growth capacity, lipid peroxidation, surface antigens on T-cells, and adherent cell suppression were observed in lymphocytes aged in vivo or in vitro. Based on these results, we suggest that blood aged in vitro may provide a simple but effective model to study many aspects of cellular aging in vivo. To further examine this hypothesis and to further understand the aging process, vitamin E was used to examine its effect on in vitro aging. The results from lymphocyte growth capacity assays show that vitamin E provides a protective effect against in vitro aging similar to that observed for in vivo aging. Examination of lymphocytes in culture allows monitoring of biochemical alterations, nutrient requirements, and other parameters in vitro, without effect on the subject. This technique therefore provides an opportunity to examine a wide variety of factors and a system eventually in which mechanisms to prevent or impede diseases can be tested.
Allostery and assembly oflac repressor protein
(1998) Barry, Jennifer Kara; Matthews, Kathleen S.
Allostery in lac repressor ligand binding is based upon the ability of the protein to assume alternate ligand-bound conformations. The X-ray crystal structures of repressor bound to different ligands indicate the end points of conformational transitions. To detect changes in the environment within the repressor upon ligand binding, single tryptophan substitutions were generated in the following locations: the N-terminus (Tyr 7); the junction between the N-terminus and the N-subdomain (Leu 62); the N-subdomain of the monomer-monomer interface (Glu 100 and Gln117); the central region of the core (His 74, Tyr273, and Phe293); the C-subdomain (Phe226); and the C-terminus (Lys325). Changes in fluorescence properties upon inducer binding are only detected for tryptophans substituted at His74, Tyr273, and Phe 293. Fluorescence properties are not altered upon operator binding. Thus, in the regions of lac repressor probed by these substitutions, the inducer-bound form differs from the conformation of the unliganded form. The roles of His74 and Asp278 in operator and inducer binding were also explored. His74 and Asp 278 form a potential salt bridge between the N- and C-subdomains in the repressor core. Mutations were generated at both positions to vary size, charge, and polarity in an effort to determine the nature and importance of this interaction. All repressor proteins produced by mutating His74 and/or Asp278 possess altered ligand binding behavior. Interestingly, only the H74W mutant repressor did not display the characteristic ∼1000-fold decrease in operator affinity when bound to inducer. The oligomeric state of lac repressor is a result of the free energy involved in both assembly and folding. The overall stability of tetrameric repressor was determined by two approaches. The free energy of complete unfolding by urea-induced denaturation of tetrameric repressor is ∼49.1 kcal/mol. The total free energy for denaturation of tetrameric repressor calculated by combining all of the assembly and folding events is ∼58.8 kcal/mol. These results demonstrate unequivocally that the tetrameric lac repressor is an extremely stable protein. However, the difference between these free energy values suggests that the same transitions may not be monitored in the two different approaches.
Biophysical and biochemical characterization of the disconnected interacting protein 1, DIP1, from Drosophila melanogaster
(2005) Catanese, Daniel James, Jr; Matthews, Kathleen S.
Transcription regulation and RNA metabolism, two essential cellular processes, are linked by Disconnected Interacting Protein 1 (DIP1) in Drosophila melanogaster. This linkage occurs through DIP1 interactions with double-stranded RNA (dsRNA) and its association with other proteins, such as Disconnected (zinc-finger transcription factor), Ultrabithorax (Ubx) (Hox transcription factor), Suppressor of variegation 3--9 (chromatin methyltransferase), and Adat (tRNA deaminase). This thesis explores the molecular mechanisms of DIP1 function by elucidating its interactions with dsRNA and two protein partners, Adat and Ubx. Proteins with dsRNA-binding domains bind dsRNA with high affinity and no sequence specificity in vitro. Nonetheless, these proteins are involved in many important facets of cellular metabolism in vivo that encompass splicing, deamination, and RNA interference. Analytical ultracentrifugation suggests that DIP1 is a homodimer in solution, consistent with data for other proteins with dsRNA-binding domains. Gel retardation demonstrates that DIP1 prefers dsRNA over dsRNA. Additionally, DIP1 binds the Adenovirus VA1 RNA with the highest affinity we have been able to identify for this class of proteins. DIP1 also binds to a stem-loop structure similar to those involved in long-range enhancer effects of the Bithorax complex as well as in the generation of two microRNAs that potentially regulate Ubx. Adat·DIP1 interaction was established in yeast two-hybrid assays, and the Adat interaction with DIP1 was confirmed with GST pulldown experiments. DIP1 binds with high affinity to the pre-tRNAala, a potential in vivo target of Adat. We hypothesize that Adat is recruited by the DIP1·RNA complex in order to properly deaminate the pre-tRNA ala. DIP1 also binds Ubx, a Hox transcription factor necessary for haltere formation during embryogenesis. All Hox proteins rely on protein-protein interactions to define their cellular function, although few such interactions have been identified. The Ubx·DIP1 interaction was demonstrated using phage display, immunoprecipitation, GST pulldown assays, and gel supershifts in complex with DNA. Yeast one-hybrid assays establish DIP1 can block Ubx activation, and a functional consequence of this interaction may be to differentiate Hox function in vivo. Elucidating these molecular mechanisms via DIP1 interactions potentially provide insight into cancer regulation and a physical bridge that links transcription and RNA metabolism.
Biophysical characterization of gold nanocrystal protein conjugates: Formation, stoichiometry and function
(2006) Calabretta, Michelle K.; Matthews, Kathleen S.
The successful application of bio/nano materials in medicine, materials science, and molecular electronics is dependent on the development of functional, well-defined hybrid materials. In this thesis, I show that the overall structure and function of protein-gold nanocrystal conjugates is influenced by protein surface charge, stoichiometry, and orientation on the nanostructure. By comparing the non-specific conjugation behavior of the lac Repressor (LacI), lysozyme, and alpha-lactalbumin, we establish that basic regions are significantly involved in the assembly of bio/nano conjugates. Super structures, such as controlled nanocrystal aggregates, can result from non-specific protein conjugation depending on the number of basic regions on the protein surface. Moreover, proteins with basic functional domains, like the DNA binding domain of LacI, pose a challenge because non-specific conjugation through these regions adversely affects biofunction. This obstacle can be avoided by specifically conjugating proteins through regions not significantly involved in function. In order to prevent conjugation through the LacI DNA binding domain, we developed a mutant with solvent exposed cysteine residues to direct conjugation to gold nanocrystals through a gold-sulfur bond. The formation and stoichiometry of LacI- and T334C-gold nanocrystal conjugates was followed by protein radiolabeling and analytical ultracentrifugation, two solution techniques that circumvent the challenges associated with spectroscopic characterization of bio/nano conjugates. These techniques provided additional confirmation that LacI conjugates through a weaker, reversible electrostatic interaction, whereas T334C conjugates are more robust, in agreement with the prediction that T334C conjugates through a non-reversible gold-sulfur bond. Lastly, the operator DNA binding function of these conjugates was assessed with nitrocellulose filter binding, analytical ultracentrifugation, and electrophoretic mobility shift. Interestingly, the order of DNA-repressor-nanocrystal complex formation had an impact on operator binding. Regardless of the order of complex formation, LacI conjugates retain little to no DNA binding function. T334C, however, retains significant operator binding if the operator-repressor complex is formed prior to conjugation. These results demonstrate that specific conjugation through regions of low functional significance can greatly improve the biofunction of conjugated proteins. This thesis provides an improved understanding of the interaction between biomolecules and nanostructures, which will benefit the design of materials that are structurally and functionally sound.
Biophysical characterization of the allosteric transition in lactose repressor protein (LacI)
(2005) Zhan, Hongli; Matthews, Kathleen S.
Allosteric transition, the basis of signal transduction and central to the function of regulatory proteins (e.g., transcriptional factors), is widely involved in biological systems with conformational change as a key characteristic. Although end-state structures are known for many proteins, less is known about the underlying detailed mechanism of the allosteric transition. We have used LacI as a model system to investigate this process at the atomic level. The work in this thesis focuses on three regions of LacI: the core pivot region, the N-subdomain monomer-monomer interface, and the hinge region. Characterization of representative mutants (L148F, S151P, P320A, and Q60G/L148F) demonstrated that the core pivot region exerts long-range effects on LacI function. For L148F and S151P, operator and inducer binding are altered in an inverse fashion with binding for one ligand strengthened, and binding for the other ligand weakened. Further characterization of L148F and S 151P has indicated that the conformational equilibrium is shifted towards the induced state in L148F and towards the repressed end in S151P. This conclusion is supported by detailed thermodynamic ligand binding assays and UV difference spectra. Detailed unfolding/refolding studies further suggest that the intrinsic ligand-binding properties of L148F and S151P are altered. Global fitting of all ligand-binding data is underway to further characterize these shifts. Our data for K84 hydrophobic variants (K84A/L) disclose impeded allosteric response to inducer, a state that is supported by a unique pattern in UV difference spectra. Operator release kinetics for K84A/L in response to IPTG suggest that two inducer molecules are required to release operator DNA. Characterization of 13 substitutions at V52, including binding to operator sequence variants, indicates a dominant role of the protein-operator interaction in LacI allostery and high affinity operator binding. Moreover, subsets of mutants that decouple inducer binding and conformational change were identified. In summary, this thesis work emphasizes the key role of several regions in LacI allostery, identifies several LacI allosteric intermediates, and discloses intermediates trapped along the allosteric pathway by mutation that correlate with points along the TMD simulation.
Chemical and spectral studies of the Lac repressor protein and its trypsin resistant core
(1982) Smith, Ann; Matthews, Kathleen S.; Palmer, Graham; Olson, John S.; Beckingham, Kathleen M.
The core protein produced by mild proteolytic digestion of the lac repressor has been purified on phosphocellulose, The repressor and core proteins were reacted with the sulfhydryl specific reagents, 2-chloromercuri-4-nitrophenol and fluorescein mercuric acetate. Modification of the cysteine residues did not alter the affinity of the proteins for inducer molecules. The operator binding activity of both proteins was unaffected by the reaction with 2-chloromercuri-4-nitrophenol; however, this binding was essentially abolished upon modification with fluorescein mercuric acetate. This loss of operator DNA binding activity in response to modification supports the thesis that determinants for specific DNA binding are located in the core region of the protein. Fluorescence spectral studies on repressor modified with 2-chloromercuri-4-nitrophenol and fluorescein mercuric acetate were performed. The quenching observed upon titration of repressor with either reagent indicates that energy transfer was occurring between the protein tryptophans and the cysteine-conjugated chromophores. Inclusion of dithiothreitol during the titration prevented the labelling of the cysteines; a corresponding decrease in energy transfer was seen. The addition of of inducer produces a blue shift in the repressor emission spectrum, but did not affect the quenching process. The quenching was sensitive to dithiothreitol for the repressor-inducer system as it had been for the repressor protein alone. The spectral behavior of the core protein was essentially identical to that displayed by the repressor.
Cloning and characterization of CMF, a density sensing factor in Dictyostelium discoideum
(1994) Jain, Renu; Matthews, Kathleen S.
Conditioned medium factor (CMF), is an 80 kD glycoprotein which is the ligand in a cell density sensing system used by developing Dictyostelium discoideum cells. CMF is slowly secreted by cells when they starve, and the extracellular level of CMF then becomes an indicator that is sensed to determine the density of starving cells. CMF cDNA has been cloned and encodes a novel 62.6 kD protein. Bacterially synthesized recombinant CMF has as much CMF activity as native CMF, indicating that glycosylation is not required for the activity. The active site of the CMF protein lies within an 88 amino acid region near the N-terminus. Immunofluorescence with affinity-purified anti-CMF antibodies indicates that CMF is present in all vegetative and developing cells. In the vegetative cells, CMF is sequestered inside previously uncharacterized vesicles and begins to be slowly secreted upon starvation. CMF antisense transformants do not aggregate unless starved in the presence of exogenous CMF. To understand how CMF regulates the aggregation of cells, the effect of CMF on cAMP signal transduction was examined. The activation of Ca$\sp{++}$ influx in response to a pulse of cAMP is strongly inhibited in cells lacking CMF and can be rescued by as little as 10 seconds exposure of cells to CMF. The activation of phospholipase C and the binding of GTP to the membranes in response to cAMP are not affected by CMF. To determine whether CMF is sensed by cell surface receptors, we examined binding of iodinated recombinant CMF to live cells. We found saturable binding to six-hour-starved cells at 3.4 $\times$ 10$\sp4$ molecules/cell with a K$\sb{\rm D}$ of 2.1 nM. The binding saturates in 30 minutes and a Scatchard plot of the binding data indicates that there is only one class of receptor. The binding can be competed with either the native endogenous CMF, recombinant CMF, or the 88 amino acid active fragment region. Very little binding is seen to vegetative cells, with maximal binding seen in cells starved for 6-8 hours. Normal levels of CMF binding are observed with CMF antisense cells, this data indicates that CMF is not required for the accumulation of its own receptor.
Density sensing in Dictyostelium discoideum: Evidence for the sequestering of a density sensing factor in the plasma membrane and a role for this factor in cell aggregation
(1992) Taphouse, Carl Richard; Matthews, Kathleen S.
A density sensing factor has been isolated from Dictyostelium discoideum and is known as conditioned media factor (CMF). CMF has been shown to cause starved vegetative Dictyostelium cells to differentiate into one of two cell types, stalk or spore cells. One interesting aspect of CMF concerns its release: CMF mRNA is present in vegetative Dictyostelium cells, yet secretion of the CMF protein only occurs during early and late development. My research reveals evidence that CMF is sequestered in the plasma membrane of vegetative cells. Further experiments show that CMF anti-sense transformants are motile but do not form cell aggregates. I suggest that CMF may have a role in chemotaxis which could involve interactions with components of the cytoskeleton. I have identified a few cytoskeletal components in vegetative and developing Dictyostelium cells which may provide useful information concerning the developmental process.
Developing Novel Protein-based Materials using Ultrabithorax: Production, Characterization, and Functionalization
(2011) Huang, Zhao; Matthews, Kathleen S.; Bondos, Sarah E.
Compared to 'conventional' materials made from metal, glass, or ceramics, protein-based materials have unique mechanical properties. Furthermore, the morphology, mechanical properties, and functionality of protein-based materials may be optimized via sequence engineering for use in a variety of applications, including textile materials, biosensors, and tissue engineering scaffolds. The development of recombinant DNA technology has enabled the production and engineering of protein-based materials ex vivo . However, harsh production conditions can compromise the mechanical properties of protein-based materials and diminish their ability to incorporate functional proteins. Developing a new generation of protein-based materials is crucial to (i) improve materials assembly conditions, (ii) create novel mechanical properties, and (iii) expand the capacity to carry functional protein/peptide sequences. This thesis describes development of novel protein-based materials using Ultrabithorax, a member of the Hox family of proteins that regulate developmental pathways in Drosophila melanogaster . The experiments presented (i) establish the conditions required for the assembly of Ubx-based materials, (ii) generate a wide range of Ubx morphologies, (iii) examine the mechanical properties of Ubx fibers, (iv) incorporate protein functions to Ubx-based materials via gene fusion, (v) pattern protein functions within the Ubx materials, and (vi) examine the biocompatibility of Ubx materials in vitro . Ubx-based materials assemble at mild conditions compatible with protein folding and activity, which enables Ubx chimeric materials to retain the function of appended proteins in spatial patterns determined by materials assembly. Ubx-based materials also display mechanical properties comparable to existing protein-based materials and demonstrate good biocompatibility with living cells in vitro . Taken together, this research demonstrates the unique features and future potential of novel Ubx-based materials.
Effect of laminar shear stress on gene regulation, protein synthesis, and protein secretion by cultured human endothelial cells
(1990) Diamond, Scott Lee; Matthews, Kathleen S.
To test the hypothesis that wall shear stress generated by blood flow may regulate endothelial cell expression of blood clot dissolving proteins or vasoactive proteins, an in vitro perfusion system was used to expose human umbilical vein endothelial cell monolayers to well defined, laminar fluid flow. Protein production studies utilized immunoassays, while semi-quantitative studies of messenger RNA levels in a small numbers of cells required a reverse transcription/polymerase chain reaction technique. Secretion by endothelial cells of the two main regulators of the fibrinolytic (ie blood clot dissolving) system, tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) were not affected by exposure to venous levels of shear stress (4 dynes/cm$\sp2$). However, at arterial shear stresses of 15 and 25 dynes/cm$\sp2$, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unstimulated by shear stress over the entire physiological range. The tPA mRNA level was many fold higher ($>$10 fold) in endothelial cells sheared for 24 hours than in stationary controls. The mRNA level of the common house-keeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was found to be the same in control and sheared cells. The fact that GAPDH was unregulated indicates selectively in cellular response to shear stress in addition to validating the PCR technique. Endothelin (ET), a 21-amino acid peptide secreted by endothelial cells, has vasoconstrictor and mitogenic activity for vascular smooth muscle cells. Fluid shear stress of 25 dynes/cm$\sp2$ caused a rapid and sustained drop in endothelin production after only 2 hours exposure to shear stress. Endothelin secretion was not affected by venous shear stress of 4 dynes/cm$\sp2$. The mRNA level for endothelin in cells exposed to shear stress was almost undetectable, indicating that the drop in protein secretion is due to a drop in transcription of the message RNA for endothelin. The mRNA level of basic fibroblast growth factor (bFGF) was found to be the same in cells sheared for 24 hours as in controls. Enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces involves transcriptional events and could explain the deposition of fibrin in low shear zones near arterial bifurcations. Flow-regulated ET expression may explain the inverse correlation of fluid shear stress with: (1) the localization of atherosclerotic lesions near vessel bifurcations and; (2) the severity of intimal hyperplasia in surgical vein bypass grafts and vessel anastomotic sites.
Evolution of the activation domain in a Hox transcription factor
(UPV/EHU Press, 2018) Liu, Ying; Huang, Annie; Booth, Rebecca M.; Mendes, Gabriela Geraldo; Merchant, Zabeena; Matthews, Kathleen S.; Bondos, Sarah E.
Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved.
Examination of the C-terminal assembly motif in lactose repressor
(1999) Wycuff, Diane Reed; Matthews, Kathleen S.
Assembly requirements for lactose repressor protein have been examined by substitutions in the leucine heptad repeat (LHR) tetramerization domain. All a and d positions of the LHR were substituted with Ile (II) or a positions with Leu and d positions with Ile (ILI). Additionally, identical substitutions made in the previously-constructed LacI/GCN4 LHR chimera, Izip were termed IzI and IzLI. To express these proteins, a system was designed employing the popular T7&phis; promoter without usual repression by lactose repressor protein. Cloning T7 gene 1 under arabinose repressor control on pBAD33 (Guzman et al., 1995) generated the plasmid pTara. Following co-transformation of pTara and expression plasmid, production of both T7 RNA polymerase and target protein was induced with L-arabinose. High expression levels of lactose repressor mutant proteins were obtained, and the general applicability of this system was confirmed using human p53 and Drosophila Ubx proteins. Proteins II and ILI demonstrated elution profiles from column chromatography typical of predominantly monomeric assembly, whereas IzI and IzLI eluted at comparable ionic strengths to wild type LacI. Gel filtration chromatography indicated that IzI is of the correct size for hexameric protein, and IzLI is in an equilibrium between tetramer and monomer. IzI and IzLI bound neither wild type operator nor completely palindromic operator sequence with specific affinity, and no decrease in operator binding was observed for either protein with inducer. IzI and IZLI bound inducer with wild type affinity, but did not exhibit the wild type reduction in affinity normally observed at pH 9.2. Trypsin digestion studies indicated that both of these proteins were reduced to lower molecular weight bands than wild type lactose repressor and that IzLI is digested to these lower weight states more rapidly than IzI. Urea denaturation studies yielded midpoints of denaturation for both proteins lower than wild type lactose repressor protein, and less steep slopes for the unfolding/dissociation transitions. These data are collectively consistent with folding to a wild type monomeric tertiary structure but anomalous assembly to dimer and higher order oligomers. The changes in dimer assembly apparently have consequences for operator recognition and cooperativity of inducer binding.
Experimental characterization and molecular dynamics simulation of the allosteric transition in the Escherichia coli lactose repressor
(2009) Xu, Jia; Matthews, Kathleen S.
The lactose repressor protein (LacI), a prototypic negative transcriptional regulator in E. coli, relies on an allosteric conformational change for its function. Targeted molecular dynamics (TMD) simulation of this LacI transition predicts that residues located in/near the inducer binding pocket, especially D149 and S193, play a critical role in the early stage of this allosteric process. Single mutants at D149 and S193, characterized by a series of biochemical and biophysical experiments, present limited information about LacI allostery. In contrast, double mutants are much more informative: D149A/S193A exhibits wild-type properties, which exclude the requirement for inter-residue hydrogen bond formation in the allosteric response. However, D149C/S193C purified from cell extracts shows decreased sensitivity to inducer binding, while retaining wild-type binding affinities for both operator and inducer. By manipulating cysteine oxidation, the more reduced state of D149C/S193C responds to inducer more similarly to wild-type protein, whereas the more oxidized state displays diminished inducer sensitivity. D149C/S193C exhibits near wild-type binding parameters for operator DNA and inducer, with comparable rate constants for binding to IPTG and dissociation from operator DNA. These features of D149C/S193C indicate that the novel disulfide bond formed in this mutant impedes the allosteric transition, consistent with the role of this region predicted by TMD simulation. V150C/V192C displays wild-type binding properties, presumably due to its reduced state. Interestingly, S151C/V192C in a partially oxidized state displays wild-type DNA and IPTG binding affinities, and retains normal response to IPTG binding. These data suggest that mobility of the entire flexible loop (residues 149-156) may not be the crucial element for Lad allosteric regulation. Further, a molecular dynamics simulation method was used to probe the motions that are necessary for the conformational change in LacI. The results of this simulation indicate that the backbone of residue 149 is the feature that may play a critical role in LacI allosteric regulation. In summary, biochemical characterization and computational simulation of multiple LacI mutants provide evidence for the functional roles of specific residues (and their interaction) and shed light on LacI allostery.
Exploration of DNA binding selectivity, transcriptional regulation, and evolutionary relationships of the Hox protein Ultrabithorax
(2007) Liu, Ying; Matthews, Kathleen S.
Hox transcription factors direct differentiation in all tissue layers and many organ systems and therefore have highly specific tissue-dependent functions. The DNA-binding homeodomain of Hox proteins can bind a wide array of nucleotide sequences with similar high affinities, suggesting regions outside the homeodomain must impact DNA interaction. Utilizing a series of deletion mutants, we identified three binding affinity modulation regions outside the homeodomain. DNA binding is inhibited ∼2-fold by the YPWM motif and microexon regions (I1) and ∼40-fold by the large disordered I2 region. High affinity binding is partially restored by the N-terminal 174 residues (R region) in a length-dependent manner. Both I2 and R regions partially overlap the Ubx transcription activation domain, allowing communication between these functional systems. Evolutionary variations in the amino acid sequences of most of these regions potentially differentiate Ubx•DNA interactions.
Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins
(American Society for Biochemistry and Molecular Biology, 2015) Bondos, Sarah E.; Swint-Kruse, Liskin; Matthews, Kathleen S.
To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: the prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection.
Functional and structural studies of influenza B virus hemagglutinin
(2013-09-16) Ni, Fengyun; Ma, Jianpeng; Tao, Yizhi Jane; Matthews, Kathleen S.
Influenza A and B viruses are major causes of seasonal flu epidemics each year. Hemagglutinin (HA) mediates the binding of virus to host cell and the fusion with host membrane. The crystal of HA in complex with antibody that reveals the mechanism by which antibody recognizes HA may not diffract to high resolution, thereby preventing the accurate interpretation of the structural model. The application of normal mode refinement that aims for improving the structure quality at the low resolution is tested. These studies provide some guidelines for future refinement of HA-antibody complex structures. By comparing the residues constituting the base of the receptor binding site of influenza A and B virus HAs, it is found that they share some similarities, except for a Phe at position 95 of influenza B virus hemagglutinin (BHA) versus Tyr in of influenza A virus hemagglutinin (AHA). The recombinant protein BHA containing the F95Y mutation exhibits the increased receptor binding affinity and specificity. However, recombinant viruses with the Phe95Tyr mutation show lower erythrocyte agglutination titer and decreased binding abilities with different cell lines. The replication of the Phe95Tyr mutant virus in mice is also attenuated. These data suggest that the increased receptor binding ability of HA alone is not advantageous to the pathogenesis of the viruses. The structure of BHA2 (a portion of BHA near the C-terminus) at the post-fusion state has been determined to 2.45 Å resolution. This protein forms a hairpin-like conformation rich in -helices. About 70 residues from the N-terminus is a three-stranded coiled coil, and the remaining of the protein packs in anti-parallel against the groove formed by the central helices. In the post-fusion state of BHA2, the helix converted from the B-loop in pre-fusion state contacts the C-terminal fragment of this protein with more hydrophobic interactions as compared to AHA2. This structure illustrates the distinct stabilization strategy employed by BHA2 to form a post-fusion state that resembles that for AHA2. These studies will further the understanding of BHA with respect to its role in receptor binding ability and fusion.
Genetic and physical studies of quaternary structure of lactose repressor
(1993) Chen, Jie; Matthews, Kathleen S.
The oligomeric assembly of the lactose repressor has been studied by a series of deletion and extension mutants at the C-terminal sequence. Deletion of the presumed mini leucine zipper sequence at the C-terminus (-5 aa, -11 aa, -18 aa, and -32 aa) has resulted in a family of dimeric repressors, while deletion of the last four amino acids (-4 aa) does not affect the oligomeric state or function of the repressor. Furthermore, extension of the C-terminus with leucine zipper-forming sequences (G359V+5 and GCN/C) has been deduced to strengthen a subunit interaction in tetramers. The role of the presumed mini leucine zipper in tetramer formation for the lac repressor is therefore confirmed. In addition, a long-axis dimer (GCN/C-Y282D) is obtained from increasing the leucine zipper length and disrupting a presumably distinctive interface composed of Tyr$\sp{282}$ and Lys$\sp{84}$. The generation of this altered protein confirms the postulate that there are two distinctive subunit interfaces for the assembly of tetrameric repressor. Detailed characterization of these mutant repressors has provided insight into the relationship between structure and function in the lac repressor. The subunit communication upon inducer-binding in a tetrameric repressor is between the two side-by-side monomers and does not appear to involve monomers in end-to-end orientation. The lower operator-affinity observed for the deletion dimers compared to the tetramer is derived from a difference in the association process.
Growth and root inhibition in Axenic hydrilla verticillata cultures
(1984) Cook, Rebecca Ann; Ward, C. H.; Tomson, Mason B.; Matthews, Kathleen S.
Hydrilla verticillata Royle is a submersed aquatic weed infesting waterways throughout the world. Hydrilla was introduced into the United States in 196 and has spread across the southern states. Hydrilla has extensive reproductive capacity, spreading by seeds, axillary and subterranean vegetative buds, fragmentation, and stolons. Subterranean buds, called tubers, are the predominant form of reproduction. This reproductive potential has made mechanical, chemical, and biological control largely ineffective. To study the life cycle of Hydrilla, Elaine and Ward (1981) developed a method for producing axenic cultures from tubers. The process by which axenic cultures were produced involves surface treatment of the tuber with sodium hypochlorite and removal of outer bud scales. Tubers were grown in a mineral salts media containing 15 ug/ml penicillin and 1 ug/ml streptomycin to prevent bacterial growth. Two percent glucose and .5 per cent casein were added to facilitate detection of bacterial contamination. Axenic cultures produced by this method have inhibited shoot and root growth. In the present study, different variables in the process were analyzed for inhibitory effects. Addition of yeast extract, IAA, IBA, GAg, and kinetin failed to overcome stem or root growth inhibition. Neither NaOCl treatment nor dissection as separate factors affected growth or root development. Sodium hypochlorite treatment followed by dissection resulted in retarded growth at times, although mature plants did develop. Addition of penicillin and streptomycin or penicillin, streptomycin, glucose, and casein resulted in inhibited growth and root production. Casein and streptomycin both inhibited stem and root growth in all cases. Glucose slightly enhanced growth. Penicillin had no significant effects on shoot growth or root formation and growth. Axenic cultures with penicillin and glucose exhibited no inhibitions. Streptomycin inhibits growth of roots at 15, 5, and 1 ug/ml. Penicillin did not affect growth of stems or roots at 25, 75, or 15 ug/ml but did not effectively control bacteria. Neomycin, ampicillin, tetracycline, and chloramphenicol were tested separately at 25, 75, and 15 ug/ml and in combinations at 75 ug/ml as possible alternatives to streptomycin. All were inhibitory to shoot growth. Root formation was significantly inhibited by all concentrations of chloramphenicol, and the higher concentrations of neomycin and tetracycline. Several of the antibiotic combinations, tested in conjunction with 25 ug/ml penicillin, were effective against bacteria but were inhibitory to hydrilla growth at tested concentrations.
In vitro mutational analysis of the inducer binding site of lactose repressor
(1994) Chang, Wen-I; Matthews, Kathleen S.
The inducer binding site of the E. coli lactose repressor has been examined by in vitro characterization of mutant proteins with amino acid substitutions at the hypothetical inducer binding site (Sams, C. F., Vyas, N. K., Quiocho, F. A. and Matthews, K. S. (1984) Nature 310, 429-430). Since the major force for sugar interaction comes from hydrogen bonding between the inducer and the hydrophilic side chains in the inducer binding site of lac repressor, Lys$\sp{84}$, Asp$\sp{88}$, Asp$\sp{130}$ and Asp$\sp{274}$ at the hypothetical inducer binding site were chosen to be substituted. Using site-directed mutagenesis, each site was substituted with four different amino acids that altered side chain length, polarity or charge. In addition, double mutations were introduced at Lys$\sp{84}$ and Tyr$\sp{282}$ sites in order to elucidate the role of Lys$\sp{84}$ in subunit interaction. Through characterizing mutant proteins at the Lys$\sp{84}$ site in vitro, including inducer binding properties, operator binding activities, immunoblotting pattern, and gel filtration behavior, we conclude that Lys$\sp{84}$ is at the same subunit interface as Tyr$\sp{282}$ and is not involved directly in the inducer binding site. A similar situation applies to Asp$\sp{88}$, which does not appear to be in the inducer binding site and does not directly contribute energy to inducer binding. The data on mutants at Asp$\sp{130}$ indicate no effect on inducer binding, while mutations at Asp$\sp{274}$ suggest an essential role is played by this amino acid. Based on the fact that mutations at the Asp$\sp{274}$ site did not show any spectroscopic alteration in the presence of inducer, even though the presence of IPTG protected iodide quenching of Trp$\sp{220}$ in the inducer binding site, we propose that this amino acid is essential for the conformational change upon inducer binding. This proposal is strongly supported by the X-ray crystallographic data obtained for periplasmic sugar binding proteins whose sequences show homology to lac repressor core domain. Thus, all the mutational data presented in this study are consistent with the recently proposed structure of lac core protein which is based on sequence alignment and molecular replacement modeling (Nichols, J. C., Vyas, N. K., Quiocho, F. A. and Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612).
Interactions between lac repressor protein and bromodeoxyuridine-substituted operator DNA: Identification of a specific amino acid-nucleotide contact using UV footprinting and crosslink formation
(1990) Wick, Kyle Lynn; Matthews, Kathleen S.
As the classical model for negative transcriptional control in prokaryotes and the subject of concentrated experimental attention, the lactose operon of Escherichia coli presents a well-defined system for studying genetic control through protein-DNA binding interactions. Binding of repressor at its cognate operator sequence within the regulatory region of the operon, while responsive to environmental conditions, efficiently inhibits transcription initiation by RNA polymerase. The high binding affinity and degree of specificity exhibited by this protein-DNA complex has encouraged investigation of the nature of the contacts formed. We have explored specific contacts between the lac repressor and operator using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40 bp operator provides an indirect means of probing the major groove of operator DNA for critical contacts between the repressors and the 5-methyl of individual thymidines. As a photoreactive species, BrdU 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 thirteen unique sites of inducer-sensitive protection along the operator sequence (+1, 2, 3, 4, 6, 8, 13, 15, 16, 18, 19, 20, 21) using this method compared to per-substitution with BrdU (Ogata and Gilbert, 1977). 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 (+3, 4, 14, 18, 19). Comparison of the UV protection results and the cross-linking data shows that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts. Using these same BrdU-substituted operator DNAs, we attempted to define individual protein-DNA interactions with respect to the specific amino acid(s) making contact at a selected site within the operator sequence. With the selection of the T$\sb{+3}$ site for our initial investigation, the cross-linked complex was formed and isolated. These polypeptide-DNA species were prepared for final analysis through a series of steps including proteolysis and anion-exchange HPLC. Protein sequence analysis on the purified peptide-operator complex identified a peptide spanning Val23 through Lys33. The data suggest His29 as the specifically crosslinked amino acid.

Browsing by Author "Matthews, Kathleen S."

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