Trimethylamine
N-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins, induces folding, and counteracts the denaturing effects of urea, pressure, and ice. To establish the ...mechanism behind these effects, isotopic substitution neutron-scattering measurements were performed on aqueous solutions of TMAO and 1:1 TMAO-urea at a solute mole fraction of 0.05. The partial pair distribution functions were extracted using the empirical potential structure refinement method. The results were compared with previous results obtained with isosteric
tert-butanol, as well as the available data from spectroscopy and molecular-dynamics simulations. In solution, the oxygen atom of TMAO is strongly hydrogen-bonded to, on average, between two and three water molecules, and the hydrogen-bond network is tighter in water than in pure water. In TMAO-urea solutions, the oxygen atom in TMAO preferentially forms hydrogen bonds with urea. This explains why the counteraction is completed at a 2:1 urea/TMAO concentration ratio, independently of urea concentration. These results strongly support models for the effect of TMAO on the stability of proteins based on a modification of the simultaneous equilibria that control hydrogen bonding between the peptide backbone and water or intramolecular sites, without any need for direct interaction between TMAO and the protein.
1. Introduction 148 2. Basics of X-ray and neutron scattering 149 2.1 Elastic scattering of electromagnetic radiation by a single electron 149 2.2 Scattering by assemblies of electrons 151 2.3 ...Anomalous scattering and long wavelengths 153 2.4 Neutron scattering 153 2.5 Transmission and attenuation 155 3. Small-angle scattering from solutions 156 3.1 Instrumentation 156 3.2 The experimental scattering pattern 157 3.3 Basic scattering functions 159 3.4 Global structural parameters 161 3.4.1 Monodisperse systems 161 3.4.2 Polydisperse systems and mixtures 163 3.5 Characteristic functions 164 4. Modelling 166 4.1 Spherical harmonics 166 4.2 Shannon sampling 169 4.3 Shape determination 170 4.3.1 Modelling with few parameters: molecular envelopes 171 4.3.2 Modelling with many parameters: bead models 173 4.4 Modelling domain structure and missing parts of high-resolution models 178 4.5 Computing scattering patterns from atomic models 184 4.6 Rigid-body refinement 187 5. Applications 190 5.1 Contrast variation studies of ribosomes 190 5.2 Structural changes and catalytic activity of the allosteric enzyme ATCase 191 6. Interactions between molecules in solution 203 6.1 Linearizing the problem for moderate interactions: the second virial coefficient 204 6.2 Determination of the structure factor 205 7. Time-resolved measurements 211 8. Conclusions 215 9. Acknowledgements 216 10. References 216 A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies – on ribosomes and on the allosteric protein aspartate transcarbamoylase – illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.
The interaction of the osmolytes trimethylamine N-oxide (TMAO) and urea in aqueous solutions at 40 °C was investigated by isotopic substitution neutron scattering at a TMAO mole fraction of 0.05 and ...TMAO/urea concentration ratios of 1 : 2 and 1 : 4. The partial pair distribution functions obtained by the empirical potential structure refinement method are consistent with those obtained previously for similar pure TMAO and 1 : 1 TMAO-urea solutions and indicate that urea progressively replaces the water molecules in the first coordination shell of the TMAO oxygen atom. The apparent association constant for the TMAO : urea complex (K(1)) was calculated to be 0.14 M(-1), which is of the same order as the experimental urea-protein binding constants per site reported in the literature. This confirms that the two osmolytes act independently at least in the physiological range.
Some matrix materials proposed for the preparation of solid lipid nanoparticles (e.g. trilaurin) are difficult to crystallize after processing by melt-homogenization. In an attempt to overcome this ...difficulty, the effect of saturated long-chain phospholipids on the crystallization of nanoparticles based on trilaurin, trimyristin, tripalmitin and tristearin was studied. The phospholipids were used as emulsifiers in combination with sodium glycocholate. Saturated phospholipids increased the crystallization temperature of the triglyceride by several degrees compared to soybean phospholipids. The crystallization pattern was more complex in such systems due to solidification of the phospholipid chains prior to triglyceride crystallization. For most triglycerides, egg lecithin also induced crystallization at higher temperatures than natural soybean lecithin. With trilaurin dispersions, the effect of phospholipids can be utilized to induce crystallization at temperatures relevant for larger scale preparation. The polymorphic transitions of the triglycerides were slower in the presence of egg and saturated lecithin leading to a higher stability of the metastable α-form. These effects were particularly pronounced in tristearin systems where a predominant fraction of α-phase particles could be observed even after long-term cold storage in dispersions containing hydrogenated soybean lecithin or DPPC. The possibility to prepare triglyceride nanoparticles stable in specific modifications offers new opportunities to study effects of polymorphic form on colloidal stability, drug loading and release properties of such dispersions.
European perch (Perca fluviatilis) are increasingly farmed as a human food source. Viral infections of European perch remain largely unexplored, thereby putting farm populations at incalculable risk ...for devastating fish epizootics and presenting a potential hazard to consumers. To address these concerns, we applied metatranscriptomics to identify disease-associated viruses in European perch farmed in Switzerland. Unexpectedly, in clinically diseased fish we detected novel freshwater fish filoviruses, a novel freshwater fish hantavirus, and a previously unknown rhabdovirus. Hantavirus titers were high, and we demonstrated virus in macrophages and gill endothelial cells by using in situ hybridization. Rhabdovirus titers in organ samples were low, but virus could be isolated on cell culture. Our data add to the hypothesis that filoviruses, hantaviruses, and rhabdoviruses are globally distributed common fish commensals, pathogens, or both. Our findings shed new light on negative-sense RNA virus diversity and evolution.
A program suite for one‐dimensional small‐angle scattering data processing running on IBM‐compatible PCs under Windows 9x/NT/2000/XP is presented. The main program, PRIMUS, has a menu‐driven ...graphical user interface calling computational modules to perform data manipulation and analysis. Experimental data in binary OTOKO format can be reduced by calling the program SAPOKO, which includes statistical analysis of time frames, averaging and scaling. Tools to generate the angular axis and detector response files from diffraction patterns of calibration samples, as well as binary to ASCII transformation programs, are available. Several types of ASCII files can be directly imported into PRIMUS, in particular, sasCIF or ILL‐type files are read without modification. PRIMUS provides basic data manipulation functions (averaging, background subtraction, merging of data measured in different angular ranges, extrapolation to zero sample concentration, etc.) and computes invariants from Guinier and Porod plots. Several external modules coupled with PRIMUSvia pop‐up menus enable the user to evaluate the characteristic functions by indirect Fourier transformation, to perform peak analysis for partially ordered systems and to find shape approximations in terms of three‐parametric geometrical bodies. For the analysis of mixtures, PRIMUS enables model‐independent singular value decomposition or linear fitting if the scattering from the components is known. An interface is also provided to the general non‐linear fitting program MIXTURE, which is designed for quantitative analysis of multicomponent systems represented by simple geometrical bodies, taking shape and size polydispersity as well as interparticle interference effects into account.
The crystallization temperature and polymorphism of tripalmitin nanoparticles in colloidal dispersions prepared by melt-homogenization and stabilized with different pharmaceutical surfactants (sodium ...glycocholate, sodium oleate, tyloxapol, Solutol HS 15, Cremophor EL) and their combinations with soybean phospholipid (Lipoid S100) were investigated to establish the influence of the emulsifiers on these parameters. There were no major effects on the crystallization temperature but remarkable differences in the time-course of polymorphic transitions after crystallization of the triglyceride particles indicate interaction between the surfactant layer and the triglyceride matrix. The metastable α-modification was most stable in dispersions solely stabilized with glycocholate. Upon fast cooling from the melt, these dispersions form an uncommon type of α-modification that displays only a very weak small-angle reflection indicating poor ordering between triglyceride layers. Slow crystallization of these glycocholate-stabilized nanoparticles yields the usual α-form. Electron microscopic investigations reveal that, in both cases, the particles in the α-modification are less anisometric than those of the stable β-form. These results indicate that major rearrangements still may take place in solid lipid nanoparticles after recrystallization.
A matter of technique: Time‐resolved X‐ray scattering was used to probe the photolysis of Ru3(CO)12 in cyclohexane, and a new intermediate was identified besides the two μ‐CO intermediates known from ...ultrafast IR spectroscopy (see scheme). The major and hitherto undetected intermediate contains only terminal CO and thus escaped detection by IR spectroscopy based on absorption bands of bridging CO.
An
ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of ...dummy residues which fits the experimental scattering pattern up to a resolution of 0.5
nm. The efficiency of the method is illustrated by the
ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.
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