Equilibrium model selection: dTTP induced R1 dimerization

<p>Abstract</p> <p>Background</p> <p>Biochemical equilibria are usually modeled iteratively: given one or a few fitted models, if there is a lack of fit or over fitting, a new model with additional or fewer parameters is then fitted, and the process is repeated. The problem with this approach is that different analysts can propose and select different models and thus extract different binding parameter estimates from the same data. An alternative is to first generate a comprehensive standardized list of plausible models, and to then fit them exhaustively, or semi-exhaustively.</p> <p>Results</p> <p>A framework is presented in which equilibriums are modeled as pairs (<it>g</it>, <it>h</it>) where <it>g </it>= 0 maps total reactant concentrations (system inputs) into free reactant concentrations (system states) which <it>h </it>then maps into expected values of measurements (system outputs). By letting dissociation constants <it>K</it>_{<it>d </it>}be either freely estimated, infinity, zero, or equal to other <it>K</it>_<it>d</it>, and by letting undamaged protein fractions be either freely estimated or 1, many <it>g </it>models are formed. A standard space of <it>g </it>models for ligand-induced protein dimerization equilibria is given. Coupled to an <it>h </it>model, the resulting (<it>g</it>, <it>h</it>) were fitted to dTTP induced R1 dimerization data (R1 is the large subunit of ribonucleotide reductase). Models with the fewest parameters were fitted first. Thereafter, upon fitting a batch, the next batch of models (with one more parameter) was fitted only if the current batch yielded a model that was better (based on the Akaike Information Criterion) than the best model in the previous batch (with one less parameter). Within batches models were fitted in parallel. This semi-exhaustive approach yielded the same best models as an exhaustive model space fit, but in approximately one-fifth the time.</p> <p>Conclusion</p> <p>Comprehensive model space based biochemical equilibrium model selection methods are realizable. Their significance to systems biology as mappings of data into mathematical models warrants their development.</p

The frontline antibiotic vancomycin induces a zinc starvation response in bacteria by binding to Zn(II).

Vancomycin is a front-line antibiotic used for the treatment of nosocomial infections, particularly those caused by methicillin-resistant Staphylococcus aureus. Despite its clinical importance the global effects of vancomycin exposure on bacterial physiology are poorly understood. In a previous transcriptomic analysis we identified a number of Zur regulon genes which were highly but transiently up-regulated by vancomycin in Streptomyces coelicolor. Here, we show that vancomycin also induces similar zinc homeostasis systems in a range of other bacteria and demonstrate that vancomycin binds to Zn(II) in vitro. This implies that vancomycin treatment sequesters zinc from bacterial cells thereby triggering a Zur-dependent zinc starvation response. The Kd value of the binding between vancomycin and Zn(II) was calculated using a novel fluorometric assay, and NMR was used to identify the binding site. These findings highlight a new biologically relevant aspect of the chemical property of vancomycin as a zinc chelator.This work was supported by funding from the Royal Society, UK (516002.K5877/ROG), the Medical Research Council, UK (G0700141). A.Z. was supported from the Said foundation and Cambridge Trust.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep1960

ENZO: A Web Tool for Derivation and Evaluation of Kinetic Models of Enzyme Catalyzed Reactions

We describe a web tool ENZO (Enzyme Kinetics), a graphical interface for building kinetic models of enzyme catalyzed reactions. ENZO automatically generates the corresponding differential equations from a stipulated enzyme reaction scheme. These differential equations are processed by a numerical solver and a regression algorithm which fits the coefficients of differential equations to experimentally observed time course curves. ENZO allows rapid evaluation of rival reaction schemes and can be used for routine tests in enzyme kinetics. It is freely available as a web tool, at http://enzo.cmm.ki.si

Northern Adriatic response to a wintertime bora wind event

The unprecedented suite of research activities that focused on the Adriatic in winter 2003 provided a multi-faceted view of bora events and the resulting oceanic response

Bora event variability and the role of air-sea feedback

Author Posting. © American Geophysical Union, 2007. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 112 (2007): C03S18, doi:10.1029/2006JC003726.A two-way interacting high resolution numerical simulation of the Adriatic Sea using the Navy Coastal Ocean Model (NCOM) and Coupled Ocean/Atmosphere Mesoscale Prediction System (COAMPS®) was conducted to improve forecast momentum and heat flux fields, and to evaluate surface flux field differences for two consecutive bora events during February 2003. (COAMPS® is a registered trademark of the Naval Research Laboratory.) The strength, mean positions and extensions of the bora jets, and the atmospheric conditions driving them varied considerably between the two events. Bora 1 had 62% stronger heat flux and 51% larger momentum flux than bora 2. The latter displayed much greater diurnal variability characterized by inertial oscillations and the early morning strengthening of a west Adriatic barrier jet, beneath which a stronger west Adriatic ocean current developed. Elsewhere, surface ocean current differences between the two events were directly related to differences in wind stress curl generated by the position and strength of the individual bora jets. The mean heat flux bias was reduced by 72%, and heat flux RMSE reduced by 30% on average at four instrumented over-water sites in the two-way coupled simulation relative to the uncoupled control. Largest reductions in wind stress were found in the bora jets, while the biggest reductions in heat flux were found along the north and west coasts of the Adriatic. In bora 2, SST gradients impacted the wind stress curl along the north and west coasts, and in bora 1 wind stress curl was sensitive to the Istrian front position and strength. The two-way coupled simulation produced diminished surface current speeds of ∼12% over the northern Adriatic during both bora compared with a one-way coupled simulation.The research support for J. Pullen, J. D. Doyle, and T. Haack was provided by the Office of Naval Research (ONR) program elements 0602435N and 0601153N

One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening†

The enzyme pteridine reductase 1 (PTR1) is a potential target for new compounds to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chemical series were identified containing aminobenzothiazole and aminobenzimidazole scaffolds, respectively. One of the hits (2-amino-6-chloro-benzimidazole) was subjected to crystal structure analysis and a high resolution crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogues (2-amino-benzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein-ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative bindingmode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, derivatives were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochemical properties were obtained

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH–rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell

Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an a-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the a-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions