Biomedical subjects

Mary A Dwyer

Publications and source records attributed to Mary A Dwyer.

6 recordsLinked to original sources

PubMed2006

Local encoding of computationally designed enzyme activity.

One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously, we showed that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here, we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10(5)-10(6)-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is encoded locally within the layer of residues directly in contact with the substrate and that, in this case, at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

Amino Acid Sequence↗

PubMed2006

Definition of the molecular basis for estrogen receptor-related receptor-alpha-cofactor interactions.

Estrogen receptor-related receptor-alpha (ERRalpha) is an orphan nuclear receptor that does not appear to require a classical small molecule ligand to facilitate its interaction with coactivators and/or hormone response elements within target genes. Instead, the apo-receptor is capable of interacting in a constitutive manner with coactivators that stimulate transcription by acting as protein ligands. We have screened combinatorial phage libraries for peptides that selectively interact with ERRalpha to probe the architecture of the ERRalpha-coactivator pocket. In this manner, we have uncovered a fundamental difference in the mechanism by which this receptor interacts with peroxisome proliferator-activated receptor-gamma coactivator-1alpha, as compared with members of the steroid receptor coactivator subfamily of coactivators. Our findings suggest that it may be possible to develop ERRalpha ligands that exhibit different pharmacological activities as a consequence of their ability to differentially regulate coactivator recruitment. In addition, these findings have implications beyond ERRalpha because they suggest that subtle alterations in the structure of the activation function-2 pocket within any nuclear receptor may enable differential recruitment of coactivators, an observation of notable pharmaceutical importance.

Amino Acid Motifs↗

PubMed2004

Computational design of a biologically active enzyme.

Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure. We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The inherent generality of the design method suggests that many enzymes can be designed by this approach.

Algorithms↗

PubMed2004

Periplasmic binding proteins: a versatile superfamily for protein engineering.

The diversity of biological function, ligand binding, conformational changes and structural adaptability of the periplasmic binding protein superfamily have been exploited to engineer biosensors, allosteric control elements, biologically active receptors and enzymes using a combination of techniques, including computational design. Extensively redesigned periplasmic binding proteins have been re-introduced into bacteria to function in synthetic signal transduction pathways that respond to extracellular ligands and as biologically active enzymes.

Computer-Aided Design↗

PubMed2003

Computational design of receptor and sensor proteins with novel functions.

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.

Amino Acid Sequence↗

PubMed2002

Construction of a fluorescent biosensor family.

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.

Amino Acid Sequence↗