Biomedical subjects

Rebecca C Wade

Publications and source records attributed to Rebecca C Wade.

10 recordsLinked to original sources

PubMed2025

Design of nanobody targeting SARS-CoV-2 spike glycoprotein using CDR-grafting assisted by molecular simulation and machine learning.

The design of proteins capable effectively binding to specific protein targets is crucial for developing therapies, diagnostics, and vaccine candidates for viral infections. Here, we introduce a complementarity-determining region (CDR) grafting approach for designing nanobodies (Nbs) that target specific epitopes, with the aid of computer simulation and machine learning. As a proof-of-concept, we designed, evaluated, and characterized a high-affinity Nb against the spike protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. The designed Nb, referred to as Nb Ab.2, was synthesized and displayed high-affinity for both the purified receptor-binding domain protein and to the virus-like particle, demonstrating affinities of 9 nM and 60 nM, respectively, as measured with microscale thermophoresis. Circular dichroism showed the designed protein's structural integrity and its proper folding, whereas molecular dynamics simulations provided insights into the internal dynamics of Nb Ab.2. This study shows that our computational pipeline can be used to efficiently design high-affinity Nbs with diagnostic and prophylactic potential, which can be tailored to tackle different viral targets.

Spike Glycoprotein, Coronavirus↗

PubMed2003

MolSurfer: A macromolecular interface navigator.

We describe the current status of the Java molecular graphics tool, MolSurfer. MolSurfer has been designed to assist the analysis of the structures and physico-chemical properties of macromolecular interfaces. MolSurfer provides a coupled display of two-dimensional (2D) maps of the interfaces generated with the ADS software and a three-dimensional (3D) view of the macromolecular structure in the Java PDB viewer, WebMol. The interfaces are analytically defined and properties such as electrostatic potential or hydrophobicity are projected on to them. MolSurfer has been applied previously to analyze a set of 39 protein-protein complexes, with structures available from the Protein Data Bank (PDB). A new application, described here, is the visualization of 75 interfaces in structures of protein-DNA and protein-RNA complexes. Another new feature is that the MolSurfer web server is now able to compute and map Poisson-Boltzmann electrostatic potentials of macromolecules onto interfaces. The MolSurfer web server is available at http://projects.villa-bosch.de/mcm/software/molsurfer.

Computer Graphics↗

PubMed2003

Implicit solvent models for flexible protein-protein docking by molecular dynamics simulation.

The suitability of three implicit solvent models for flexible protein-protein docking by procedures using molecular dynamics simulation is investigated. The three models are (i) the generalized Born (GB) model implemented in the program AMBER6.0; (ii) a distance-dependent dielectric (DDD) model; and (iii) a surface area-dependent model that we have parameterized and call the NPSA model. This is a distance-dependent dielectric model modified by neutralizing the ionizable side-chains and adding a surface area-dependent solvation term. These solvent models were first tested in molecular dynamics simulations at 300 K of the native structures of barnase, barstar, segment B1 of protein G, and three WW domains. These protein structures display a range of secondary structure contents and stabilities. Then, to investigate the performance of the implicit solvent models in protein docking, molecular dynamics simulations of barnase/barstar complexation, as well as PIN1 WW domain/peptide complexation, were conducted, starting from separated unbound structures. The simulations show that the NPSA model has significant advantages over the DDD and GB models in maintaining the native structures of the proteins and providing more accurate docked complexes.

Bacterial Proteins↗

PubMed2003

DSMM: a Database of Simulated Molecular Motions.

We describe a Database of Simulated Molecular Motions (DSMM). This database is designed to serve as a single searchable site for locating movies and animations from simulations of biomolecules. DSMM is accessible via a webserver at: http://projects.villa-bosch.de/mcm/database/dsmm.

Computer Graphics↗

PubMed2003

Concerted simulations reveal how peroxidase compound III formation results in cellular oscillations.

A major problem in mathematical modeling of the dynamics of complex biological systems is the frequent lack of knowledge of kinetic parameters. Here, we apply Brownian dynamics simulations, based on protein three-dimensional structures, to estimate a previously undetermined kinetic parameter, which is then used in biochemical network simulations. The peroxidase-oxidase reaction involves many elementary steps and displays oscillatory dynamics important for immune response. Brownian dynamics simulations were performed for three different peroxidases to estimate the rate constant for one of the elementary steps crucial for oscillations in the peroxidase-oxidase reaction, the association of superoxide with peroxidase. Computed second-order rate constants agree well with available experimental data and permit prediction of rate constants at physiological conditions. The simulations show that electrostatic interactions depress the rate of superoxide association with myeloperoxidase, bringing it into the range necessary for oscillatory behavior in activated neutrophils. Such negative electrostatic steering of enzyme-substrate association presents a novel control mechanism and lies in sharp contrast to the electrostatically-steered fast association of superoxide and Cu/Zn superoxide dismutase, which is also simulated here. The results demonstrate the potential of an integrated and concerted application of structure-based simulations and biochemical network simulations in cellular systems biology.

Animals↗

PubMed2002

Comparative binding energy (COMBINE) analysis of OppA-peptide complexes to relate structure to binding thermodynamics.

The periplasmic oligopeptide binding component (OppA) of the oligopeptide permease found in Gram-negative bacteria acts as a receptor for peptide transport across the cell membrane and is a potential target for antibacterial drug design. OppA exhibits broad specificity, binding to diverse peptides of 2-5 amino acid residues length. Crystallographic and calorimetric measurements have been carried out by Tame et al. of the binding of 28 peptides of sequence K-X-K to OppA, where X is a natural or nonnatural amino acid. Despite this extensive experimental characterization, a clear relationship between structural and thermodynamic parameters could not be readily identified, with a complicating factor being the observation of varying numbers of water molecules at the binding interface in the different complexes. Consequently, we have applied COMparative BINding Energy (COMBINE) analysis to derive quantitative structure-activity relationships (QSARs) for these 28 OppA-tripeptide complexes. This is the first application of COMBINE analysis to predict binding enthalpies and entropies, and predictive QSAR models were obtained for these quantities as well as for binding free energies. These QSAR models highlight several protein residues and bound water molecules in the binding site, as well as the electrostatic desolvation energies of the protein and the peptides, as responsible for most of the differences in binding thermodynamics between the peptides studied. The QSAR models aid rationalization of the determinants of binding affinity of the OppA:peptide complexes and provide guides for further ligand design. This study also points to the general applicability of COMBINE analysis to estimating thermodynamic parameters for protein-peptide complexes.

Bacterial Proteins↗

PubMed2002

Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin.

Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.

Amino Acid Sequence↗

PubMed2002

Comparison of the dynamics of substrate access channels in three cytochrome P450s reveals different opening mechanisms and a novel functional role for a buried arginine.

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.

Arginine↗

PubMed2002

Biomolecular diffusional association.

The primary method for the computational study of biomolecular diffusional association is Brownian dynamics. Recent work has seen advances in the efficiency of computing association rates and in the accuracy of simulation models. New areas to which Brownian dynamics has been applied include protein polymerisation and protein adsorption to a surface. There has recently been particularly intense study of protein-protein association, and Brownian dynamics, together with other theoretical and experimental approaches, has led to new insights into the determinants of protein-protein binding kinetics.

Actins↗

PubMedDate not supplied

Elusive recognition determinants for ubiquitination.

How are proteins recognized as substrates for ubiquitination? Here we summarize insights from recent experiments that address this issue. These highlight the diversity and complexity of determinants for substrate recognition, and raise many questions for further investigation.

Animals↗