Assessing the reconstruction of macromolecular assemblies with toleranced models

We introduce toleranced models (TOMs), a generic and versatile framework meant to handle models of macromolecular assemblies featuring uncertainties on the shapes and the positions of proteins. A TOM being a continuum of nested shapes, the inner (resp. outer) ones representing high (low) confidence regions, we present topological and geometric statistics assessing features of this continuum at multiple scales. While the topological statistics qualify contacts between instances of protein types and complexes involving prescribed protein types, the geometric statistics scale the geometric accuracy of these complexes. We validate the TOM framework on recent average models of the entire nuclear pore complex (NPC) obtained from reconstruction by data integration, and confront our quantitative analysis against experimental findings related to complexes of the NPC, namely the Y-complex, the T-complex, and the Nsp1-Nup82-Nup159 complex. In the three cases, our analysis bridges the gap between global qualitative models of the entire NPC, and atomic resolution models or putative models of the aforementioned complexes. In a broader perspective, the quantitative assessments provided by the TOM framework should prove instrumental to implement a virtuous loop "model reconstruction-model selection", in the context of reconstruction by data integration.     

Improved data visualization techniques for analyzing macromolecule structural changes

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color‐blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three‐index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Prediction of ice content in biological model solutions when frozen under high pressure

High pressure is, at least, as effective as cryoprotective agents (CPAs) and are used for decreasing both homogenous nucleation and freezing temperatures. This fact gives rise to a great variety of possible cryopreservation processes under high pressure. They have not been optimized yet, since they are relatively recent and are mainly based on the pressure–temperature phase diagram of pure water. Very few phase diagrams of biological material are available under pressure. This is owing to the lack of suitable equipment and to the difficulties encountered in carrying out the measurements. Different aqueous solutions of salt and CPAs as biological models are studied in the range of 0°C down to ‐35°C, 0.1 up to 250 MPa, and 0–20% w/w total solute concentration. The phase transition curves of glycerol and of sodium chloride with either glycerol or sucrose in aqueous solutions are determined in a high hydrostatic pressure vessel. The experimental phase diagrams of binary solutions were well described by a third‐degree polynomial equation. It was also shown that Robinson and Stokes' equation at high pressure succeeds in predicting the phase diagrams of both binary and ternary solutions. The solute cryoconcentration and the ice content were calculated as a function of temperature and pressure conditions during the freezing of a binary solution. This information should provide a basis upon which high‐pressure cryopreservation processes may be performed and the damages derived from ice formation evaluated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Singlet oxygenation in microemulsion catalysed by vanadium chloroperoxidase

Non-ionic microemulsions compatible with the enzyme vanadium chloroperoxidase were designed to perform singlet oxygenation of apolar substrates. The media were based on mono- and polydisperse ethoxylated fatty alcohols (C_iE_j), octane and aqueous buffer. “Fish” diagrams were determined to identify the Winsor-boundaries and to formulate a monophasic Winsor IV microemulsion with a minimal surfactant concentration, ensuring less singlet oxygen (¹O₂) loss than in an aqueous system, thus creating a high oxygenation efficiency. The enzyme was shown to be fully stable in the microemulsion for at least 10 h, converting H₂O₂ into a constant flow of ¹O₂ in the aqueous microdomains. Part of the ¹O₂ diffuses into the organic compartments prior to fast physical deactivation of ¹O₂ by water molecules. In the apolar domains ¹O₂ quantitatively converts the model substrate 9,10-dimethylanthracene into its corresponding endoperoxide. Near-IR chemiluminescence measurements confirm that the ¹O₂ signal in the microemulsion is higher than in simple aqueous buffer. In a well-stirred (water/octane) biphasic system endoperoxide formation is also observed but the conversion rate is much lower, most likely due to stronger physical quenching of ¹O₂.     

有机概念图在黄酮类物质研究中的应用

计算了108种常见黄酮类物质的无机值/有机值比(I/O值),并就其理化性质、生理活性与其在有机概念图中分布的相关性进行了讨论。     

Liquid-ordered phases induced by cholesterol: A compendium of binary phase diagrams

Mixtures of phospholipids with cholesterol are able to form liquid-ordered phases that are characterised by short-range orientational order and long-range translational disorder. These L_o-phases are distinct from the liquid-disordered, fluid L_α-phases and the solid-ordered, gel L_β-phases that are assumed by the phospholipids alone. The liquid-ordered phase can produce spatially separated in-plane fluid domains, which, in the form of lipid rafts, are thought to act as platforms for signalling and membrane sorting in cells. The areas of domain formation are defined by the regions of phase coexistence in the phase diagrams for the binary mixtures of lipid with cholesterol. In this paper, the available binary phase diagrams of lipid-cholesterol mixtures are all collected together. It is found that there is not complete agreement between different determinations of the phase diagrams for the same binary mixture. This can be attributed to the indirect methods largely used to establish the phase boundaries. Intercomparison of the various data sets allows critical assessment of which phase boundaries are rigorously established from direct evidence for phase coexistence.     

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.     

Attempts to rationalize protein crystallization using relative crystallizability

Protein crystal growth (PCG) remains the bottleneck of crystallography despite many decades of study. The nucleation zone in the two-dimensional-phase diagram has been used to evaluate the relative crystallizability of proteins, which is expressed as a percentage over the phase area delineated by experimental protein and precipitating agent concentration ranges. For protein-salts which are subject to a direct temperature effect on solubility, as represented by Egg Lysozyme, a decrease in temperature augments the nucleation zone percentage whereas for those with retrograde solubility as a function of temperature, for example fructose-1,6-bisphosphatase in the presence and absence of AMP, an increase in temperature can significantly enhance the relative crystallizability. These results have been confirmed by the number of "hits" using PEGs as precipitating agents in Sparse Matrix Screen experiments for different proteins and are in excellent agreement with the relative crystallizability. The relationship between solubility dependence, relative crystallizability and crystallization success, has been evidenced. Such crystallizability can become a guide to identify efficient crystallization regions, providing a rational approach to PCG and structural biology.