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Bulk Solvent (bulk + solvent)
Selected AbstractsIn-house characterization of protein powderJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2010Christian Grundahl Hartmann X-ray powder diffraction patterns of lysozyme and insulin were recorded on a standard in-house powder diffractometer. The experimental powder diffraction patterns were compared with patterns calculated from Protein Data Bank coordinate data. Good agreement was obtained by including straightforward corrections for background, unit-cell parameters, disordered bulk solvent and geometric factors. In particular the solvent correction was found crucial for a good agreement. A revised Lorentz factor was derived, which gave a minor, but significant, improvement to the fit in the low-angle region. An attempt to include calculated H-atom positions did not improve the overall fit and was abandoned. The method devised was shown to be a quick and convenient tool for distinguishing precipitates and polymorphs of proteins. [source] The mechanism of alkaline hydrolysis of amides: a comparative computational and experimental study of the hydrolysis of N -methylacetamide, N -methylbenzamide, and acetanilideJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 6 2009Diana Cheshmedzhieva Abstract Theoretical computations and experimental kinetic measurements were applied in studying the mechanistic pathways for the alkaline hydrolysis of three secondary amides: N -methylbenzamide, N -methylacetamide, and acetanilide. Electronic structure methods at the HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) levels of theory are employed. The energies of the stationary points along the reaction coordinate were further refined via single point computations at the MP2/6-31+G(d,p) and MP2/6-311++G(2d,2p) levels of theory. The role of water in the reaction mechanisms is examined. The theoretical results show that in the cases of N -methylbenzamide and N -methylacetamide the process is catalyzed by an ancillary water molecule. The influence of water is further assessed by predicting its role as bulk solvent. The alkaline hydrolysis process in aqueous solution is characterized by two distinct free energy barriers: the formation of a tetrahedral adduct and its breaking to products. The results show that the rate-determining stage of the process is associated with the second transition state. The entropy terms evaluated from theoretical computations referring to gas-phase processes are significantly overestimated. The activation barriers for the alkaline hydrolysis of N -methylbenzamide and acetanilide were experimentally determined. Quite satisfactory agreement between experimental values and computed activation enthalpies was obtained. Copyright © 2008 John Wiley & Sons, Ltd. [source] Characterization of solvatochromic probes: simulation of merocyanine 540 absorption spectra in binary solvent mixtures and pure solvent systemsJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 3 2004S. Y. Bae Abstract An empirical extension of the continuum model was used to reproduce the absorption spectrum of the optical probe merocyanine 540 in numerous solvents based on 27 probe-specific parameters in conjunction with the dielectric constant and refractive index of the solvent. The calibrated absorption spectrum of this dye allowed the accurate determination of the dielectric constant and refractive index of the bulk solvent. This study incorporates several binary solvent mixtures in addition to several pure solvents of differing functionality, including protic and aprotic solvents. A single, generally applicable, set of probe-specific parameters is presented. The accuracy of the determined solvent properties using this general set of probe parameters suggests that the influence of specific solvent,solute interactions on the absorption spectrum of this dye must be constant if not insignificant in the range of solvents studied, with the notable exception of water. Copyright © 2004 John Wiley & Sons, Ltd. [source] The Complex of Apomyoglobin with the Fluorescent Dye Coumarin 153,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2004P. K. Chowdhury ABSTRACT Understanding a protein's dielectric response requires both a theoretical model and a well-defined experimental system. The former has already been proposed by Song (J. Chem. Phys. 116, 9359 [2002]). We suggest that the latter is provided by the complex of coumarin 153 (C153) with apomyoglobin (ApoMb). C153 has been exhaustively studied and has proven to be an excellent probe of the solvation dynamics of polar solvents. Myoglobin is one of the most thoroughly studied proteins. Myoglobins from a wide range of species have been subject to X-ray structural analysis and site-directed mutagenesis. Here, we demonstrate the existence of a robust C153-apomyglobin system by means of molecular dynamics simulations, equilibrium binding studies using a Job's plot and capillary electrophoresis, circular dichroism and time-resolved fluorescence. The reorganization energy of C153 bound to ApoMb is compared with that of C153 in bulk solvent using the method of Jordanides et al. (J. Phys. Chem. B 103, 7995 [1999]). [source] Buried water molecules in helical transmembrane proteinsPROTEIN SCIENCE, Issue 2 2008Robert Renthal Abstract Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules. [source] Protein dynamics control proton transfer from bulk solvent to protein interior: A case study with a green fluorescent proteinPROTEIN SCIENCE, Issue 7 2005Anoop M. Saxena Abstract The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o -nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO3, and K2SO4, a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein. [source] Determination of enzyme mechanisms by molecular dynamics: Studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenasePROTEIN SCIENCE, Issue 8 2004Swarnalatha Y. Reddy Abstract Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH, [,HC5(O,) ,C4( = O) ,] and PQQH [,HC5(OH) ,C4( = O) ,] converts to the hydroquinone PQQH2 [,C5(OH) = C4(OH) ,]. The main focus is on MD structures of MDH , PQQ , methanol, MDH , PQQH,, MDH , PQQH, sGDH , PQQ , glucose, sGDH , PQQH, (glucolactone, and sGDH , PQQH. The reaction PQQ , PQQH, occurs with Glu 171,CO2, and His 144,Im as the base species in MDH and sGDH, respectively. The general-base-catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen-bonding to the C5 = O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297,COOH would act as a proton donor for the reaction PQQH, , PQQH, if formed by transfer of the proton from Glu 171,COOH to Asp 297,CO2, in MDH. For PQQH , PQQH2, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen-bonded to Glu 171,CO2, in MDH and by Wat89 in sGDH). [source] Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domainsPROTEIN SCIENCE, Issue 5 2002Michal Otyepka Abstract One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the ,4,-helix-loop-,4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels. [source] On the use of low-resolution data for translation search in molecular replacementACTA CRYSTALLOGRAPHICA SECTION A, Issue 1 2002Andrei Fokine Low-resolution reflections (approximately 15,Å and lower) are very useful for the translation search in molecular replacement because they are less sensitive to model errors compared with the traditionally used reflections of resolution 4,10,Å. At low resolution, however, the contribution from the bulk solvent is quite significant and corresponding structure factors calculated from a macromolecular model cannot be compared with experimental values if this contribution is neglected. The proposed method provides a way of fast translation searches where low-resolution reflections are taken into account. Test calculations using several experimental data sets show a dramatic improvement in the signal after the bulk-solvent correction and low-resolution reflections were included in the calculation; this improvement allowed unambiguous identification of the solution. [source] A smooth and differentiable bulk-solvent model for macromolecular diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010T. D. Fenn Inclusion of low-resolution data in macromolecular crystallography requires a model for the bulk solvent. Previous methods have used a binary mask to accomplish this, which has proven to be very effective, but the mask is discontinuous at the solute,solvent boundary (i.e. the mask value jumps from zero to one) and is not differentiable with respect to atomic parameters. Here, two algorithms are introduced for computing bulk-solvent models using either a polynomial switch or a smoothly thresholded product of Gaussians, and both models are shown to be efficient and differentiable with respect to atomic coordinates. These alternative bulk-solvent models offer algorithmic improvements, while showing similar agreement of the model with the observed amplitudes relative to the binary model as monitored using R, Rfree and differences between experimental and model phases. As with the standard solvent models, the alternative models improve the agreement primarily with lower resolution (>6,Å) data versus no bulk solvent. The models are easily implemented into crystallographic software packages and can be used as a general method for bulk-solvent correction in macromolecular crystallography. [source] Bias in cross-validated free R factors: mitigation of the effects of non-crystallographic symmetryACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2006Felcy Fabiola Current methods of free R factor cross-validation assume that the structure factors of the test and working sets are independent of one another. This assumption is only an approximation when the modeled structure occupies anything less than the full asymmetric unit. Through progressive elimination of reflections from the working set, starting with those expected to be most correlated to the test set, small biases in free R can be measured, presumably because of over-sampling of the Fourier transform owing to bulk solvent in the crystal. This level of bias may be of little practical importance, but it rises to significant levels with increasing non-crystallographic symmetry owing to wider correlations between structure factors than hitherto appreciated. In the presence of 15-fold non-crystallographic symmetry, with resolutions commonly attainable in macromolecular crystallography, it may not be possible to calculate an unbiased free R factor. Methods are developed for the calculation of reduced-bias free R factors through elimination of the strongest correlations between test and working sets. With 180-fold non-crystallographic symmetry they may not be an accurate indicator of absolute quality, but they do yield the correct optimal weighting for stereochemical restraints. [source] The role of solvent transport in cryo-annealing of macromolecular crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Douglas H. Juers Macromolecular crystals are usually cooled to ,100,K for X-ray diffraction experiments in order to diminish lattice damage arising from the ionizing radiation. Such cooling often produces lattice disorder, but this disorder can sometimes be substantially reduced by cycling the crystal between low and higher temperatures (called annealing). Here, two related aspects of cryocooling and annealing are investigated using crystals of ,-galactosidase and thermolysin. Firstly, as has been reported with other systems, there is an optimal cryoprotectant concentration above and below which diffraction is poor, with high mosaicity, diffuse scatter and low signal to noise. Measurements of the bulk density of the respective cryosolvents are consistent with the idea that at the optimal cryoprotectant concentration the contraction of the bulk solvent on cooling largely compensates for the contraction of the macromolecular lattice. Secondly, by controlling the relative humidity of the gas that contacts the crystal during the high (room) temperature phase, it is found that water is either imported into or exported out of the crystals during the melting phase of annealing. This water transport appears to change the concentration of the cryoprotectant solution and in so doing alters its thermal contraction. Thus, annealing appears to be involved, at least in part, in the tuning of the thermal contraction of the bulk solvent to best compensate for lattice contraction. Furthermore, it is found that if the cryoprotectant concentration is initially too high then annealing is more successful than if the concentration is initially too low. This result suggests that the search for optimal cryoprotectant conditions may be facilitated by equilibration of the crystal to relatively high cryoprotectant concentration followed by annealing. [source] Flat bulk-solvent model: obtaining optimal parametersACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2002Andrei Fokine A bulk-solvent correction is regularly used for macromolecular refinement. The flat model of the bulk solvent is considered to be the most reliable. It is shown that the standard procedure does not always result in the optimal values of the bulk-solvent correction parameters. A method to obtain the best values for parameters ksol and Bsol of the flat-solvent model is discussed. The values of correctly determined parameters for crystallographic structures deposited in the Protein Data Bank are clustered around ksol = 0.35,e,Å,3 and Bsol = 46,Å2, which have a reasonable physical meaning. Such a distribution allows the use of these mean values of solvent parameters for many practical applications when refined parameters cannot be obtained, especially when an atomic model in the unit cell is not yet known. [source] | ||||||||||