Triton X－100对紫膜蛋白的效应

本文采用同步辐射小角Ｘ射线散射方法研究了用非离子表面活性剂ＴｒｉｔｏｎＸ—１００处理后的嗜盐菌紫膜及其视紫红质蛋白结构的变化。实验结果表明,用不同浓度的ＴｒｉｔｏｎＸ—１００处理紫膜碎片时,紫膜及其蛋白所处的状态有着很大变化。     

The role of water in the reversibility of thermal denaturation of lysozyme in solid and liquid states

Although unfolding of protein in the liquid state is relatively well studied, its mechanisms in the solid state, are much less understood. We evaluated the reversibility of thermal unfolding of lysozyme with respect to the water content using a combination of thermodynamic and structural techniques such as differential scanning calorimetry, synchrotron small and wide-angle X-ray scattering (SWAXS) and Raman spectroscopy. Analysis of the endothermic thermal transition obtained by DSC scans showed three distinct unfolding behaviors at different water contents. Using SWAXS and Raman spectroscopy, we investigated reversibility of the unfolding for each hydration regime for various structural levels including overall molecular shape, secondary structure, hydrophobic and hydrogen bonding interactions. In the substantially dehydrated state below 37 wt% of water the unfolding is an irreversible process and can be described by a kinetic approach; above 60 wt% the process is reversible, and the thermodynamic equilibrium approach is applied. In the intermediate range of water contents between 37 wt% and 60 wt%, the system is phase separated and the thermal denaturation involves two processes: melting of protein crystals and unfolding of protein molecules. A phase diagram of thermal unfolding/denaturation in lysozyme - water system was constructed based on the experimental data.     

Integrative structural modeling with small angle X-ray scattering profiles

Recent technological advances enabled high-throughput collection of Small Angle X-ray Scattering (SAXS) profiles of biological macromolecules. Thus, computational methods for integrating SAXS profiles into structural modeling are needed more than ever. Here, we review specifically the use of SAXS profiles for the structural modeling of proteins, nucleic acids, and their complexes. First, the approaches for computing theoretical SAXS profiles from structures are presented. Second, computational methods for predicting protein structures, dynamics of proteins in solution, and assembly structures are covered. Third, we discuss the use of SAXS profiles in integrative structure modeling approaches that depend simultaneously on several data types.     

Sulfmyoglobin conformational change: A role in the decrease of oxy-myoglobin functionality

This work is focused at understanding the interaction of H₂S with Myoglobin (Mb), in particular the Sulfmyoglobin (SMb) product, whose physiological role is controversial and not well understood. The scattering curves, Guinier, Kratky, Porod and P(r) plots were analyzed for oxy-Mb and oxy-Hemoglobin I (oxyHbI) in the absence and presence of H₂S, using Small and Wide Angle X-ray Scattering (SAXS/WAXS) technique. Three dimensional models were also generated from the SAXS/WAXS data. The results show that SMb formation, produced by oxyMb and H₂S interaction, induces a change in the protein conformation where its envelope has a very small cleft and the protein is more flexible, less rigid and compact. Based on the direct relationship between Mb's structural conformation and its functionality, we suggest that the conformational change observed upon SMb formation plays a contribution to the protein decrease in O₂ affinity and, therefore, on its functionality.     

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s⁰_20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R_G values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.     

Lysozyme crystal growth, as observed by small angle X-ray scattering, proceeds without crystallization intermediates

A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles. Received: 28 July 1997 / Revised version: 4 December 1997 / Accepted: 5 December 1997     

A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints

We present the implementation of a target function based on Small Angle Scattering data (Gabel et al. Eur Biophys J 35(4):313-327, 2006) into the Crystallography and NMR Systems (CNS) and demonstrate its utility in NMR structure calculations by simultaneous application of small angle scattering (SAS) and residual dipolar coupling (RDC) restraints. The efficiency and stability of the approach are demonstrated by reconstructing the structure of a two domain region of the 31 kDa nuclear export factor TAP (TIP-associated protein). Starting with the high resolution X-ray structures of the two individual TAP domains, the translational and orientational domain arrangement is refined simultaneously. We tested the stability of the protocol against variations of the SAS target parameters and the number of RDCs and their uncertainties. The activation of SAS restraints results in an improved translational clustering of the domain positions and lifts part of the fourfold degeneracy of their orientations (associated with a single alignment tensor). The resulting ensemble of structures reflects the conformational space that is consistent with the experimental SAS and RDC data. The SAS target function is computationally very efficient. SAS restraints can be activated at different levels of precision and only a limited SAS angular range is required. When combined with additional data from chemical shift perturbation, paramagnetic relaxation enhancement or mutational analysis the SAS refinement is an efficient approach for defining the topology of multi-domain and/or multimeric biomolecular complexes in solution based on available high resolution structures (NMR or X-ray) of the individual domains.     

Structural Studies of an Anti-Inflammatory Lectin from Canavalia boliviana Seeds in Complex with Dimannosides

Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.     

Solution structure analysis of cytoplasmic domain of podocyte protein Neph1 using small/wide angle x-ray scattering (SWAXS)

Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 Å, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD·ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys-761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into the molecular structure and function of Neph1-CD.     

Citrate-silver nanoparticles and their impact on some environmental beneficial fungi

Colloidal suspensions of silver nanoparticles (AgNPs) with surface modified by capping with citrate ions were synthesized by chemical reduction method. Transmission and Scanning Electron Microscopy as well as darkfield Optical Microscopy provided information on the nanoparticle morphology, with fine symmetrical grains and log-normal fitted size distribution. Small Angle X-ray Scattering method allowed theoretical confirmation of colloidal silver nanoparticle fine granularity, based on measurements in the native fluid sample. UV–Vis spectrophotometry allowed studying the Localized Surface Plasmon Resonance band versus the stability of the citrate-AgNP sample after storage and after UV-C exposure. The colloidal AgNP impact on Phanerochaete chrysosporium environmental microorganisms was studied by specific biochemical investigations. Silver released from the colloidal suspension of AgNPs was supposed to induce changes in some antioxidant enzymes and in some enzymes of Krebs’ cycle. Catalase activity was moderately changed (an increase with over 50%) as well as superoxide dismutase activity, while the diminution of the activities of four dehydrogenases synthesized in the fungus mycelium was emphasized also: a decrease with about 60% for malate dehydrogenase, with over 50% for isocitrate dehydrogenase and succinate dehydrogenase and with about 40% for alpha-ketoglutarate dehydrogenase. These findings suggested the nano-toxicological issues of citrate-AgNPs impact on the environmental beneficial microorganisms.     

ISPyB: an information management system for synchrotron macromolecular crystallography

MOTIVATION: Individual research groups now analyze thousands of samples per year at synchrotron macromolecular crystallography (MX) resources. The efficient management of experimental data is thus essential if the best possible experiments are to be performed and the best possible data used in downstream processes in structure determination pipelines. Information System for Protein crystallography Beamlines (ISPyB), a Laboratory Information Management System (LIMS) with an underlying data model allowing for the integration of analyses down-stream of the data collection experiment was developed to facilitate such data management. RESULTS: ISPyB is now a multisite, generic LIMS for synchrotron-based MX experiments. Its initial functionality has been enhanced to include improved sample tracking and reporting of experimental protocols, the direct ranking of the diffraction characteristics of individual samples and the archiving of raw data and results from ancillary experiments and post-experiment data processing protocols. This latter feature paves the way for ISPyB to play a central role in future macromolecular structure solution pipelines and validates the application of the approach used in ISPyB to other experimental techniques, such as biological solution Small Angle X-ray Scattering and spectroscopy, which have similar sample tracking and data handling requirements.     

Interaction of a new anticancer prodrug, gemcitabine-squalene, with a model membrane : Coupled DSC and XRD study

Gemcitabine is an anticancer nucleoside analogue active against a wide variety of solid tumors. However it is rapidly deaminated to an inactive metabolite, leading to short biological half-life and induction of resistance. A new prodrug of gemcitabine, coupling squalene to gemcitabine (GemSq), has been designed to overcome the above drawbacks. It has been previously shown that this prodrug displays significantly higher anticancer activity than gemcitabine against leukemia. In the present study the structural modifications of dipalmitoylphosphatidylcholine (DPPC) model membranes induced by increasing concentrations of GemSQ have been investigated using small and wide angle X-ray scattering (SWAXS) and differential scanning calorimetry (DSC). At room temperature an unusual inverse bicontinuous cubic phase formed over a broad composition range. The basic bilayer structure displayed an intermediate order between those of the gel and fluid phases of DPPC. A reversible transition to a fluid lamellar phase occurred upon heating. The transitions between these two phases were governed by different mechanisms depending on the GemSq concentration in the membrane. Finally, the biological relevance of these observations for the cytotoxic activity of GemSq has been discussed.     

Low-angle X-ray scattering analysis of the Thermoplasma acidophilum nucleoprotein subunit

A DNA-protein complex isolated from Thermoplasma acidophilum has been examined using low-angle X-ray scattering measurements. In agreement with the results of electron-microscopic studies a diamter of 5.5 nm is deduced. Finally, a simplified model of the DNA-protein particles is discussed postulating a kinked DNA.     

Structural changes in alpha-crystallin and whole eye lens during heating, observed by low-angle X-ray diffraction

Whole eye lens and alpha-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 degrees C to 70 degrees C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 degrees C to 45 degrees C but increased at 50 degrees C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of alpha-crystallin gels at near-physiological concentration (approximately 300 mg/ml), so it is reasonable to assume that the alpha-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens alpha-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the alpha-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 degrees C to 45 degrees C, followed by an accelerated increase from 45 degrees C to 70 degrees C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for alpha-crystallin solutions at a variety of lower concentrations.     

The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (R_g) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.     

Structural Insights on Two Hypothetical Secretion Chaperones from <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv<Emphasis Type="Italic">. citri</Emphasis>

Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.     

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes.     

Kidney stone nano-structure — Is there an opportunity for nanomedicine development?

BackgroundKidney stone analysis techniques are well-established in the field of materials characterization and provide information for the chemical composition and structure of a sample. Nanomedicine, on the other hand, is a field with an increasing rate of scientific research, a big budget and increasingly developing market. The key scientific question is if there is a possibility for the development of a nanomedicine to treat kidney stones.Major conclusionsThe main calculi characterization techniques such as X-ray Diffraction and Fourier Transform Infrared Spectroscopy can provide information about the composition of a kidney stone but not for its nanostructure. On the other hand, Small Angle X-ray Scattering and Nitrogen Porosimetry can show the nanostructural parameters of the calculi. The combination of the previously described parameters can be used for the development of nano-drugs for the treatment of urolithiasis, while no such nano-drugs exist yet.General significanceIn this study, we focus on the most well-known techniques for kidney stone analysis, the urolithiasis management and the search for possible nanomedicine for the treatment of kidney stone disease. We combine the results from five different analysis techniques in order to represent a three dimensional model and we propose a hypothetical nano-drug with gold nanoparticles. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.     

Small-angle X-ray study on the quaternary structure of erythrocruorin from Helisoma trivolvis

The erythrocruorin from the aquatic snail Helisoma trivolvis was studied in sodium phosphate buffer at pH 6.7 by small angle X-ray scattering. The following molecular parameters were determined: radius of gyration 9.4 ± 0.1 nm and maximum dimension 29 ± 1 nm. A model which fits the experimental data well is presented. The overall shape is best described by a slightly ellipsoidal shape with a hole in the centre. A model consisting of 12 subunits forming a slightly ellipsoidal shape fits very well all scattering data.