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Kathleen S Matthews

Publications and source records attributed to Kathleen S Matthews.

11 recordsLinked to original sources
PubMed2006

Ligand interactions with lactose repressor protein and the repressor-operator complex: the effects of ionization and oligomerization on binding.

Specific interactions between proteins and ligands that modify their functions are crucial in biology. Here, we examine sugars that bind the lactose repressor protein (LacI) and modify repressor affinity for operator DNA using isothermal titration calorimetry and equilibrium DNA binding experiments. High affinity binding of the commonly-used inducer isopropyl-beta,D-thiogalactoside is strongly driven by enthalpic forces, whereas inducer 2-phenylethyl-beta,D-galactoside has weaker affinity with low enthalpic contributions. Perturbing the dimer interface with either pH or oligomeric state shows that weak inducer binding is sensitive to changes in this distant region. Effects of the neutral compound o-nitrophenyl-beta,D-galactoside are sensitive to oligomerization, and at elevated pH this compound converts to an anti-inducer ligand with slightly enhanced enthalpic contributions to the binding energy. Anti-inducer o-nitrophenyl-beta,D-fucoside exhibits slightly enhanced affinity and increased enthalpic contributions at elevated pH. Collectively, these results both demonstrate the range of energetic consequences that occur with LacI binding to structurally-similar ligands and expand our insight into the link between effector binding and structural changes at the subunit interface.

Bacterial Proteins↗
PubMed2006

Physical and genetic interactions link hox function with diverse transcription factors and cell signaling proteins.

Positional information provided by Hox homeotic transcription factors is integrated with other transcription factors and cell signaling cascades in specific combinations to dictate context- and gene-specific Hox activity. Protein-protein interactions between these groups have long been hypothesized to modulate Hox functions, yielding a context-specific function. However, difficulties in applying interaction screens to potent transcription factors have limited partner identification. A yeast two-hybrid screen using transcription activation-deficient mutants of the Drosophila melanogaster Hox protein Ultrabithorax IB identified an array of interacting proteins, consisting primarily of transcription factors and components of cell signaling pathways. Interactions were confirmed with wild-type Ultrabithorax (UBX) in phage display experiments and by immunoprecipitation for a subset of partners. In vivo assays demonstrated that two Ultrabithorax IB partners, Armadillo, regulated by Wingless/WNT signaling, and the homeodomain protein Aristaless, inhibit UBX-dependent haltere development from the default wing development pathway. Therefore, transcription factors and cell signaling proteins that subdivide Hox-specified tissues can both alter Hox function in vivo and interact with the corresponding Hox protein in vitro. UBX may also modulate partner function: the pupal death phenotype induced by ectopic expression of the UBX partner Hairy required the presence of UBX. Thus, Hox.transcription factor complexes may integrate a variety of positional cues, generating the specificity and versatility required for context-dependent Hox function.

Amino Acid Sequence↗
PubMed2005

The experimental folding landscape of monomeric lactose repressor, a large two-domain protein, involves two kinetic intermediates.

To probe the experimental folding behavior of a large protein with complex topology, we created a monomeric variant of the lactose repressor protein (MLAc), a well characterized tetrameric protein that regulates transcription of the lac operon. Purified MLAc is folded, fully functional, and binds the inducer isopropyl beta-d-thiogalactoside with the same affinity as wild-type LacI. Equilibrium unfolding of MLAc induced by the chemical denaturant urea is a reversible, apparent two-state process (pH 7.5, 20 degrees C). However, time-resolved experiments demonstrate that unfolding is single-exponential, whereas refolding data indicate two transient intermediates. The data reveal the initial formation of a burst-phase (tau < ms) intermediate that corresponds to approximately 50% of the total secondary-structure content. This step is followed by a rearrangement reaction that is rate-limited by an unfolding process (tau approximately 3 s; pH 7.5, 20 degrees C) and results in a second intermediate. This MLAc intermediate converts to the native structure (tau approximately 30 s; pH 7.5, 20 degrees C). Remarkably, the experimental folding-energy landscape for MLAc is in excellent agreement with theoretical predictions using a simple topology-based C(alpha)-model as presented in a companion article in this issue.

Bacterial Proteins↗
PubMed2005

Characterization of the folding landscape of monomeric lactose repressor: quantitative comparison of theory and experiment.

Recent theoretical/computational studies based on simplified protein models and experimental investigation have suggested that the native structure of a protein plays a primary role in determining the folding rate and mechanism of relatively small single-domain proteins. Here, we extend the study of the relationship between protein topology and folding mechanism to a larger protein with complex topology, by analyzing the folding process of monomeric lactose repressor (MLAc) computationally by using a Gō-like C(alpha) model. Next, we combine simulation and experimental results (see companion article in this issue) to achieve a comprehensive assessment of the folding landscape of this protein. Remarkably, simulated kinetic and equilibrium analyses show an excellent quantitative agreement with the experimental folding data of this study. The results of this comparison show that a simplified, completely unfrustrated C(alpha) model correctly reproduces the complex folding features of a large multidomain protein with complex topology. The success of this effort underlines the importance of synergistic experimental/theoretical approaches to achieve a broader understanding of the folding landscape.

Bacterial Proteins↗
PubMed2005

Analytical ultracentrifugation for characterizing nanocrystals and their bioconjugates.

Analytical ultracentrifugation (AU) provides a general way to probe the polydispersity of nanoparticles and the formation of bioconjugates in solution. Unconjugated gold nanocrystals show sedimentation coefficient distributions that are in agreement with size distributions as measured by TEM. AU is sensitive to the size/shape changes elicited by conjugation, in this case to lactose repressor (LacI). AU data reveal saturating protein concentrations for conjugates that correspond to the measured stoichiometry of the complex under these conditions.

Algorithms↗
PubMed2004

Hox transcription factor ultrabithorax Ib physically and genetically interacts with disconnected interacting protein 1, a double-stranded RNA-binding protein.

The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0-12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx.DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx.DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated.

Animals↗
PubMed2003

Perturbation from a distance: mutations that alter LacI function through long-range effects.

Allosteric modification of ligand binding is central to LacI transcription control. Recently, the conformational change between LacI operator- and inducer-bound states was simulated with targeted molecular dynamics (TMD) [Flynn, T. C., Swint-Kruse, L., Kong, Y., Booth, C., Matthews, K. S., and Ma, J. (2003) Protein Sci., 12, 2523-2541]. Atomic-level analyses of TMD results indicate the structural importance of the core pivot region that connects the N- and C-subdomains flanking the inducer-binding site. Further, a number of LacI mutations in the core pivot have been identified recently by their altered behaviors in phenotypic screens. Biochemical characterization of three of these variants-L148F, S151P, and P320A-provides an opportunity to directly explore the role of the core pivot in repressor function. For L148F, inducer IPTG binding affinity is strengthened, whereas O(1) operator DNA binding is diminished approximately 30-fold. In contrast, O(1) binding is increased for S151P, whereas IPTG binding is decreased. UV-difference spectroscopy and urea denaturation indicate long-range effects in both variants. Interestingly, P320A binds to DNA approximately 4-fold more tightly than wild-type, yet inducer binding is unaffected. To examine linkage between the core pivot and DNA binding domains, the L148F substitution was combined with Q60G, a previously known mutant with enhanced operator affinity. The double mutant exhibits the properties of both parent proteins, resulting in near wild-type DNA binding affinity and enhanced inducer sensitivity. These features may render Q60G/L148F more cost-effective in technological applications than wild-type repressor. As a group, the behaviors of the core pivot mutants are consistent with the allosteric structural role predicted for this region by TMD and reflect the significant long-range impact that single substitutions can elicit on protein function.

Amino Acid Substitution↗
PubMed2003

Allosteric transition pathways in the lactose repressor protein core domains: asymmetric motions in a homodimer.

The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain beta-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; pi-stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74rsquo;. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data.

Allosteric Regulation↗
PubMed2002

Transcription activation by ultrabithorax Ib protein requires a predicted alpha-helical region.

Characterization of their transcription activation domains is critical to understanding functional specificity within the Hox family of proteins. However, few Hox activation domains have been identified and none characterized in detail. In this study, promotor-reporter assays in yeast and Drosophila S2 cell culture were used to refine the boundaries of the activation domain of the Drosophila Hox protein Ultrabithorax (Ubx) and to identify critical elements within this domain. We found that residues 159-242 were sufficient for 50% function, and full transactivation capacity was achieved with inclusion of additional N-terminal sequences. Activation domain sequence and placement relative to the homeodomain differ between Ubx and other Hox proteins, consistent with the possibility that diverse activation mechanisms contribute to functional distinctions in vivo. The essential residues 159-242 in the UbxIb activation domain are predicted to contain a beta-sheet segment followed by an alpha-helix. This putative alpha-helical region was established to be necessary, but not sufficient, for transcriptional activation. Disruption of the helix by proline substitutions abolished activation function, while alteration of side chains presented on the surface of this putative helix with alanine or lysine mutations had no significant effect on activity. Collectively, these data indicate that this secondary structural element is a key component in forming an effective activation domain in the UbxIb protein. Interestingly, the alpha-helix critical for transcriptional activation is found only for Ubx orthologs from flies and not other species. The mutant Ubx proteins generated in this study have potential applications in deciphering Hox functions in vivo.

Amino Acid Sequence↗
PubMedDate not supplied

DNA binding to protein-gold nanocrystal conjugates.

The E. coli DNA binding protein lac repressor (LacI) and a derivative with a designed thiol (T334C) were developed as gold nanocrystal conjugates to assess the effects of conjugation on DNA binding function. The designed derivative was engineered with a solvent-accessible thiol to promote oriented conjugation, avoiding obstruction of the DNA-binding domain by the nanocrystal. Analytical ultracentrifugation (AU) and electrophoretic mobility shift assays (EMSA) were used to evaluate the ability of conjugated repressors to bind the natural operator DNA sequence O(1). The results show that LacI does not retain significant DNA binding function when conjugated to gold nanocrystals, presumably because the basic DNA-binding domain is the site for nonspecific conjugation. T334C, with the potential for both directed and nonspecific conjugation, shows enhanced interaction with O(1) when conjugated. Interestingly, the order of component addition is a key factor in producing functional lac repressor conjugates.

Bacterial Proteins↗