Biomedical subjects

Carol Beth Post

Publications and source records attributed to Carol Beth Post.

12 recordsLinked to original sources

PubMed2006

Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions.

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.

Algorithms↗

PubMed2006

B-myc: N-terminal recognition of myc binding proteins.

B-Myc is an endogenous, N-terminal homologue of transcription factor c-Myc that lacks the C-terminal DNA binding and protein dimerization domain of c-Myc. Clinical mutations in the c-Myc N-terminal region, and the subsequent misregulation of Myc, are implicated in the development of numerous human cancers. Myc functions to both activate and repress transcription by associating with multiple binding partners. We investigated the structural and dynamical properties of B-Myc, free or associated with the transactivation inhibitor, MM-1, and the activator, TBP, using NMR spectroscopy. B-Myc has no persistent tertiary structure, yet regions corresponding to Myc homology boxes 1 and 2 (MBI and MBII, respectively) have molten globule-like characteristics. B-Myc binds to MM-1 in a specific manner without becoming highly structured. The local regions of B-Myc involved in binding differ for MM-1 and TBP, and regions not identified by mutagenesis are found to be involved in MM-1 binding. The results provide new insights into Myc N-terminal protein-protein interactions. We propose a model for Myc regulation through differential involvement of MBI and MBII in the binding of Myc interacting proteins.

Amino Acid Sequence↗

PubMed2006

Computational and mutational analysis of human glutaredoxin (thioltransferase): probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis.

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.

Amino Acid Sequence↗

PubMed2006

Src kinase activation: A switched electrostatic network.

Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an alpha-helix (alphaC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with alphaC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.

Catalytic Domain↗

PubMed2005

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase.

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.

Animals↗

PubMed2005

Dissociation of an antiviral compound from the internal pocket of human rhinovirus 14 capsid.

WIN antiviral compounds bind human rhinovirus, as well as enterovirus and parechovirus, in an internal cavity located within the viral protein capsid. Access to the buried pocket necessitates deviation from the average viral protein structure identified by crystallography. We investigated the dissociation of WIN 52084 from the pocket in human rhinovirus 14 by using an adiabatic, biased molecular dynamics simulation method. Multiple dissociation trajectories are used to characterize the pathway. WIN 52084 exits between the polypeptide chain near the ends of betaC and betaH in a series of steps. Small, transient packing defects in the protein are sufficient for dissociation. A number of torsion-angle transitions of the antiviral compound are involved, which suggests that flexibility in antiviral compounds is important for binding. It is interesting to note that dissociation is associated with an increase in the conformational fluctuations of residues never in direct contact with WIN 52084 over the course of dissociation. These residues are N-terminal residues in the viral proteins VP3 and VP4 and are located in the interior of the capsid near the icosahedral 5-fold axis. The observed changes in dynamics may be relevant to structural changes associated with virion uncoating and its inhibition by antiviral compounds.

Antiviral Agents↗

PubMed2005

Association of sindbis virus capsid protein with phospholipid membranes and the E2 glycoprotein: implications for alphavirus assembly.

A late stage in assembly of alphaviruses within infected cells is thought to be directed by interactions between the nucleocapsid and the cytoplasmic domain of the E2 protein, a component of the viral E1/E2 glycoprotein complex that is embedded in the plasma membrane. Recognition between the nucleocapsid protein and the E2 protein was explored in solution using NMR spectroscopy, as well as in binding assays using a model phospholipid membrane system that incorporated a variety of Sindbis virus E2 cytoplasmic domain (cdE2) and capsid protein constructs. In these binding assays, synthetic cdE2 peptides were reconstituted into phospholipid vesicles to simulate the presentation of cdE2 on the inner leaflet of the plasma membrane. Results from these binding assays showed a direct interaction between a peptide containing the C-terminal 16 amino acids of the cdE2 sequence and a Sindbis virus capsid protein construct containing amino acids 19-264. Additional experiments that probed the sequence specificity of this cdE2-capsid interaction are also described. Further binding assays demonstrated an interaction between the 19-264 capsid protein and artificial vesicles containing neutral or negatively charged phospholipids, while capsid protein constructs with N-terminal truncations displayed either little or no affinity for such vesicles. The membrane-binding property of the capsid protein suggests that the membrane may play an active role in alphavirus assembly. The results are consistent with an assembly process involving an initial membrane association, whereby an association with E2 glycoprotein further enhances capsid binding to facilitate membrane envelopment of the nucleocapsid for budding. Collectively, these experiments elucidate certain requirements for the binding of Sindbis virus capsid protein to the cytoplasmic domain of the E2 glycoprotein, a critical event in the alphavirus maturation pathway.

Alphavirus↗

PubMed2004

Solution structure of dengue virus capsid protein reveals another fold.

Dengue virus is responsible for approximately 50-100 million infections, resulting in nearly 24,000 deaths annually. The capsid (C) protein of dengue virus is essential for specific encapsidation of the RNA genome, but little structural information on the C protein is available. We report the solution structure of the 200-residue homodimer of dengue 2 C protein. The structure provides, to our knowledge, the first 3D picture of a flavivirus C protein and identifies a fold that includes a large dimerization surface contributed by two pairs of helices, one of which has characteristics of a coiled-coil. NMR structure determination involved a secondary structure sorting approach to facilitate assignment of the intersubunit nuclear Overhauser effect interactions. The dimer of dengue C protein has an unusually high net charge, and the structure reveals an asymmetric distribution of basic residues over the surface of the protein. Nearly half of the basic residues lie along one face of the dimer. In contrast, the conserved hydrophobic region forms an extensive apolar surface at a dimer interface on the opposite side of the molecule. We propose a model for the interaction of dengue C protein with RNA and the viral membrane that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.

Amino Acid Sequence↗

PubMed2003

Adhesive-cohesive model for protein compressibility: an alternative perspective on stability.

As a dynamic property of folded proteins, protein compressibility provides important information about the forces that govern structural stability. We relate intrinsic compressibility to stability by using molecular dynamics to identify a molecular basis for the variation in compressibility among globular proteins. We find that excess surface charge accounts for this variation not only for the proteins simulated by molecular dynamics but also for a larger set of globular proteins. This dependence on charge distribution forms the basis for an adhesive-cohesive model of protein compressibility in which attractive forces from solvent compete with tertiary interactions that favor folding. Further, a newly recognized correlation between compressibility and the heat capacity of unfolding infers a link between compressibility and the enthalpy of unfolding. This linkage, together with the adhesive-cohesive model for compressibility, leads to the conclusion that folded proteins can gain enthalpic stability from a uniform distribution of charged atoms, as opposed to partitioning charge to the protein surface. Whether buried charged groups can be energetically stabilizing is a fundamental, yet controversial, question regarding protein structure. The analysis reported here implies that one mechanism to gain enthalpic stability involves positioning charge inside the protein in an optimal structural arrangement.

Biochemical Phenomena↗

PubMed2003

Exchange-transferred NOE spectroscopy and bound ligand structure determination.

The exchange-transferred nuclear Overhauser effect of NMR spectroscopy provides information on small-molecule ligands in association with high-molecular-weight proteins or nucleic acids, or with biomolecular assemblies such as membranes. The method has proved particularly useful for the structural analysis of proton-rich, flexible ligands and for screening mixtures of ligands for binding activity. Recent analysis has established the accuracy of bound peptide structures determined from transferred nuclear Overhauser effect data and that intermolecular spin diffusion effects do not diminish the reliability of the structural result. New applications of the method involve systems of greater complexity, such as membrane-bound receptors and ribosomes. In addition, new experiments have been developed that exploit the transfer of other types of NMR signal (saturation, cross-correlation, dipolar coupling) to obtain structural information.

Binding Sites↗

PubMed2002

Docking multiple conformations of a flexible ligand into a protein binding site using NMR restraints.

A method is described for docking a large, flexible ligand using intra-ligand conformational restraints from exchange-transferred NOE (etNOE) data. Numerous conformations of the ligand are generated in isolation, and a subset of representative conformations is selected. A crude model of the protein-ligand complex is used as a template for overlaying the selected ligand structures, and each complex is conformationally relaxed by molecular mechanics to optimize the interaction. Finally, the complexes were assessed for structural quality. Alternative approaches are described for the three steps of the method: generation of the initial docking template; selection of a subset of ligand conformations; and conformational sampling of the complex. The template is generated either by manual docking using interactive graphics or by a computational grid-based search of the binding site. A subset of conformations from the total number of peptides calculated in isolation is selected based on either low energy and satisfaction of the etNOE restraints, or a cluster analysis of the full set. To optimize the interactions in the complex, either a restrained Monte Carlo-energy minimization (MCM) protocol or a restrained simulated annealing (SA) protocol were used. This work produced 53 initial complexes of which 8 were assessed in detail. With the etNOE conformational restraints, all of the approaches provide reasonable models. The grid-based approach to generate an initial docking template allows a large volume to be sampled, and as a result, two distinct binding modes were identified for a fifteen-residue peptide binding to an enzyme active site.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2002

Intermolecular relaxation has little effect on intra-peptide exchange-transferred NOE intensities.

Exchange-transferred nuclear Overhauser enhancement (etNOE) provides a useful method for determining the 3dimensional structure of a ligand bound to a high-molecular-weight complex. Some concern about the accuracy of such structures has arisen because indirect relaxation can occur between the ligand and macromolecule. Such indirect relaxation, or spin diffusion, would lead to errors in interproton distances used as restraints in structure determination. We address this concern by assessing the extent of intermolecular spin diffusion in nineteen peptide-protein complexes of known structure. Transferred NOE intensities were simulated with the program CORONA (Calculated OR Observed NOESY Analysis) using the rate-matrix approach to include contributions from indirect relaxation between protein-ligand and intraligand proton pairs. Intermolecular spin diffusion contributions were determined by comparing intensities calculated with protonated protein to those calculated with fully deuterated protein. The differences were found to be insignificant overall, and to diminish at short mixing times and high mole ratios of peptide to protein. Spin diffusion between the peptide ligand and the protein contributes less to the etNOE intensities and alters fewer cross peaks than the well-studied intramolecular spin diffusion effects. Errors in intraligand interproton distances due to intermolecular relaxation effects were small on average and can be accounted for with the restraint functions commonly used in NMR structure determination methods. In addition, a rate-matrix approach to calculate distances from etNOESY intensities using a volume matrix comprising only intraligand intensities was found to give accurate values. Based on these results, we conclude that structures determined from etNOESY data are no less accurate due to spin diffusion than structures determined from conventional NOE intensities.

Ligands↗