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Structural Biology (structural + biology)
Selected AbstractsTextbook of Structural Biology, by Anders Liljas, Lars Liljas, Jure Piskur, Göran Lindblom, Poul Nissen, and Morten KjeldgaardPROTEIN SCIENCE, Issue 12 2009Michael Rossmann No abstract is available for this article. [source] Biomolecular Crystallography: Principles, Practice, and Applications to Structural Biology.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010By Bernhard Rupp. First page of article [source] A procedure for setting up high-throughput nanolitre crystallization experiments.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2005Crystallization workflow for initial screening, automated storage, imaging, optimization Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100,nl of protein plus 100,nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294,K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277,K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported. [source] PDB_REDO: automated re-refinement of X-ray structure models in the PDBJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2009Robbie P. Joosten Structural biology, homology modelling and rational drug design require accurate three-dimensional macromolecular coordinates. However, the coordinates in the Protein Data Bank (PDB) have not all been obtained using the latest experimental and computational methods. In this study a method is presented for automated re-refinement of existing structure models in the PDB. A large-scale benchmark with 16,807 PDB entries showed that they can be improved in terms of fit to the deposited experimental X-ray data as well as in terms of geometric quality. The re-refinement protocol uses TLS models to describe concerted atom movement. The resulting structure models are made available through the PDB_REDO databank (http://www.cmbi.ru.nl/pdb_redo/). Grid computing techniques were used to overcome the computational requirements of this endeavour. [source] Subtype-selective targeting of voltage-gated sodium channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2009Steve England Voltage-gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well-precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype-selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high-throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three-dimensional structure of voltage-gated ion channels, bringing with it the opportunity to do real structure-based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure-based drug design in the not too distant future. [source] In vivo Engineering of Tissues: Biological Considerations, Challenges, Strategies, and Future DirectionsADVANCED MATERIALS, Issue 32-33 2009V. Prasad Shastri Abstract Moving forward materials-based regenerative medicine faces many challenges to ensure clinical success. Many of these challenges lie at the interface of molecular/structural biology and materials science. This review discusses this issue from a biological and material view-point, highlighting key biological processes and variables that can impact the repair processes. From a materials design stand point, developing materials that can promote healing over scarring is the key. All indicators suggest that polymeric materials are most well-suited for de novo engineering of tissues. In addition to biomolecular signals that are involved in controlling the fate of cells and neo-tissue morphogenesis at the site of implantation, this review also discusses recent advances in design of highly functional injectable biomaterials, that show promise in controlling local biological processes. [source] The fallacies of hope: will we discover new antibiotics to combat pathogenic bacteria in time?FEMS MICROBIOLOGY REVIEWS, Issue 6 2006Miguel Vicente Abstract While newly developed technologies have revolutionized the classical approaches to combating infectious diseases, the difficulties associated with developing novel antimicrobials mean that these technologies have not yet been used to introduce new compounds into the market. The new technologies, including genomics and structural biology, open up exciting possibilities for the discovery of antibiotics. However, a substantial effort to pursue research, and moreover to incorporate the results into the production chain, is required in order to bring new antimicrobials to the final user. In the current scenario of emerging diseases and the rapid spread of antibiotic resistance, an active policy to support these requirements is vital. Otherwise, many valuable programmes may never be fully developed for lack of "interest" and funds (private and public). Will we react in time to avoid potential disaster? [source] Enzymatic deconstruction of xylan for biofuel productionGCB BIOENERGY, Issue 1 2009DYLAN DODD Abstract The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. [source] Nuclear microenvironments and cancerJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Gary S. Stein Nucleic acids and regulatory proteins are architecturally organized in nuclear microenvironments. The compartmentalization of regulatory machinery for gene expression, replication and repair, is obligatory for fidelity of biological control. Perturbations in the organization, assembly and integration of regulatory machinery have been functionally linked to the onset and progression of tumorigenesis. The combined application of cellular, molecular, biochemical and in vivo genetic approaches, together with structural biology, genomics, proteomics and bioinformatics, will likely lead to new approaches in cancer diagnostics and therapy. J. Cell. Biochem. 104: 1949,1952, 2008. © 2008 Wiley-Liss, Inc. [source] Overview and new developments in softer X-ray (2Å < , < 5Å) protein crystallographyJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2004John R. Helliwell New methodologies with synchrotron radiation and X-ray free electron lasers (XFELs) in structural biology are being developed. Recent trends in harnessing softer X-rays in protein crystallography for phase determination are described. These include reference to a data-collection test at 2.6 Å wavelength with a lysozyme crystal on SRS station 7.2 (Helliwell, 1983) and also use of softer X-rays (2,Å wavelength) to optimise f," at the xenon L1 absorption edge in the Single Isomorphous Replacement Optimised Anomalous Scattering ('SIROAS') structure determination of apocrustacyanin A1 with four, partially occupied, xenon atoms (Cianci et al., 2001; Chayen et al., 2000). The hand of the protein was determined using the f," enhanced sulphur anomalous signal from six disulphides in the protein dimer of 40,kDa. In a follow-up study the single wavelength xenon L1 -edge f," optimised data set alone was used for phase determination and phase improvement by solvent flattening etc. (CCP4 DM) (Olczak et al., 2003). Auto-tracing of the protein was feasible but required additional diffraction data at higher resolution. This latter could be avoided in future by using improved tilted detector settings during use of softer X-rays, i.e. towards back-scattering recording (Helliwell, 2002). The Olczak et al. study has already led to optimisation of the new SRS beamline MPW,MAD,10 (see www.nwsgc.ac.uk) firstly involving the thinning of the beryllium windows as much as possible and planning for a MAR Research tilted detector `desk top beamline' geometry. Thus the use of softer, i.e. 2 to 3,Å wavelength range, X-rays will allow optimisation of xenon and iodine L -edge f," and enhancing of sulphur f," signals for higher throughput protein crystallography. Softer X-rays utilisation in protein crystallography includes work done on SRS bending-magnet station 7.2 in the early 1980s by the author as station scientist (Helliwell, 1984). In the future development of XFELs these softer X-ray wavelengths could also be harnessed and relax the demands to some extent on the complexity and cost of an XFEL. Thus, by use of say 4,Å XFEL radiation and use of a back-scattering geometry area detector the single molecule molecular transform could be sampled to a spatial resolution of 2,Å, sufficient, in principle, for protein model refinement (Miao et al., 1999). Meanwhile, Miao et al. (2003) report the first experimental recording of the diffraction pattern from intact Escherichia coli bacteria using coherent X-rays, with a wavelength of 2,Å, at a resolution of 30,nm and a real-space image constructed. The new single-particle X-ray diffraction-imaging era has commenced. [source] A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: In vivo characterization and structure/function relationshipsMASS SPECTROMETRY REVIEWS, Issue 4 2008Arjen Scholten Abstract Mass spectrometry has evolved in recent years to a well-accepted and increasingly important complementary technique in molecular and structural biology. Here we review the many contributions mass spectrometry based studies have made in recent years in our understanding of the important cyclic nucleotide activated protein kinase A (PKA) and protein kinase G (PKG). We both describe the characterization of kinase isozymes, substrate phosphorylation, binding partners and post-translational modifications by proteomics based methodologies as well as their structural and functional properties as revealed by native mass spectrometry, H/D exchange MS and ion mobility. Combining all these mass spectrometry based data with other biophysical and biochemical data has been of great help to unravel the intricate regulation of kinase function in the cell in all its magnificent complexity. © 2008 Wiley Periodicals, Inc. Mass Spec Rev 27: 331,353, 2008 [source] Biochemical applications of mass spectrometry in pharmaceutical drug discoveryMASS SPECTROMETRY REVIEWS, Issue 3 2005Kieran F. Geoghegan Abstract Biochemical applications of mass spectrometry (MS) are important in the pharmaceutical industry. They comprise compositional analyses of biomolecules, especially proteins, and methods that measure molecular functions such as ligand binding. In early drug discovery, MS is used to characterize essential reagents and in structural biology. A number of MS-based methods have been proposed for use in high-throughput screening (HTS), but are unlikely to supplant established radiometric and fluorometric methods for this purpose. These methods, which include pulsed-ultrafiltration MS, frontal affinity chromatography-MS, and size-exclusion chromatography-MS, may ultimately be most successful in the post-screening lead development phase. In full development, MS is used heavily in the search for biomarkers that can be used to gauge disease progression and drug action. This review gives equal attention to the technical aspects of MS-based methods and to selective pressures present in the industrial environment that influence their chances of gaining wide application. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:347,366, 2005 [source] 2004 ASM Conference on the New Phage Biology: the ,Phage Summit'MOLECULAR MICROBIOLOGY, Issue 5 2005Sankar Adhya Summary In August, more than 350 conferees from 24 countries attended the ASM Conference on the New Phage Biology, in Key Biscayne, Florida. This meeting, also called the Phage Summit, was the first major international gathering in decades devoted exclusively to phage biology. What emerged from the 5 days of the Summit was a clear perspective on the explosive resurgence of interest in all aspects of bacteriophage biology. The classic phage systems like , and T4, reinvigorated by structural biology, bioinformatics and new molecular and cell biology tools, remain model systems of unequalled power and facility for studying fundamental biological issues. In addition, the New Phage Biology is also populated by basic and applied scientists focused on ecology, evolution, nanotechnology, bacterial pathogenesis and phage-based immunologics, therapeutics and diagnostics, resulting in a heightened interest in bacteriophages per se, rather than as a model system. Besides constituting another landmark in the long history of a field begun by d'Herelle and Twort during the early 20th century, the Summit provided a unique venue for establishment of new interactive networks for collaborative efforts between scientists of many different backgrounds, interests and expertise. [source] Nitric oxide in plants: the history is just beginningPLANT CELL & ENVIRONMENT, Issue 3 2001M. V. Beligni ABSTRACT Nitric oxide (NO) is a bioactive molecule that exerts a number of diverse activities in phylogenetically distant species, as well as opposing effects in related biological systems. It was firstly described in mammals as a major messenger in the cardiovascular, immune and nervous system, in which it plays regulatory, signalling, cytoprotective and cytotoxic effects (Ignarro, Annual Review of Pharmacology and Toxicology 30, 535,560, 1990; Anbar, Experientia 51, 545,550, 1995). This versatility is mainly achieved through interactions with targets via either a redox or an additive chemistry (Stamler, Cell 78, 931,936, 1994). For this reason, metal- and thiol-containing proteins serve as major target sites for NO: these include signalling proteins, receptors, enzymes, transcription factors and DNA, among others. Furthermore, NO is a small, highly diffusible molecule. It rapidly crosses biological membranes and triggers various different processes in a short period of time. In this context, NO can co-ordinate and regulate cellular functions of microsomes and organelles such as mitochondria. The ubiquity of NO reactions, as well as the finding that the biochemical and molecular mechanisms underlying many physiological processes are well conserved between diverse species, have opened the exploration of NO chemistry in different organisms. Among these, plants were not the exception. The research in plants has been focused on three main fields: (i) the search for NO or any source of NO generation; (ii) the examination of the effects of NO upon exogenous treatments; and (iii) the search for the same molecules involved in NO-sensitive transduction pathways as in animals (e.g. cGMP, Ca2+, calmodulin). As it is evident from this review, recent progress on NO functionality in plants has been impressive. With the use of biochemistry, molecular genetics and structural biology, together with classical physiological approaches, an explosion of new discoveries will surely begin. It is certainly a good time for plant biologists. [source] G-protein-coupled receptor structures were not built in a dayPROTEIN SCIENCE, Issue 7 2009Tracy M. Blois Abstract Among the most exciting recent developments in structural biology is the structure determination of G-protein-coupled receptors (GPCRs), which comprise the largest class of membrane proteins in mammalian cells and have enormous importance for disease and drug development. The GPCR structures are perhaps the most visible examples of a nascent revolution in membrane protein structure determination. Like other major milestones in science, however, such as the sequencing of the human genome, these achievements were built on a hidden foundation of technological developments. Here, we describe some of the methods that are fueling the membrane protein structure revolution and have enabled the determination of the current GPCR structures, along with new techniques that may lead to future structures. [source] Backbone structure of a small helical integral membrane protein: A unique structural characterizationPROTEIN SCIENCE, Issue 1 2009Richard C. Page Abstract The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three-dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C-terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 Å rmsd backbone structure with a transmembrane domain defined at 0.40 Å. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long-range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins. [source] Comparison of binding energies of SrcSH2-phosphotyrosyl peptides with structure-based prediction using surface area based empirical parameterizationPROTEIN SCIENCE, Issue 10 2000Denise A. Henriques Abstract The prediction of binding energies from the three-dimensional (3D) structure of a protein,ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent-accessible surface area-based calculations for the prediction of the binding of Src-SH2 domain with a series of tyrosyl phosphopeptides based on the high-affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+3 position has been changed. Two other peptides based on the C-terminal regulatory site of the Src protein and the platelet-derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area-based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (,Cp, ,S, ,H, and ,G) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent-accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters; however, the tolerance of this approach is not sufficient to make it ubiquitously applicable. [source] Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2003Laetitia Cravello The combination of hydrogen exchange and mass spectrometry has been widely used in structural biology, providing views on protein structure and protein dynamics. One of the constraints is to use proteases working at low pH and low temperature to limit back-exchange during proteolysis. Although pepsin works in these conditions and is currently used in such experiments, sequence coverage is not always complete especially for large proteins, and the spatial resolution of the exchange rate is limited by the size of the resulting peptides. In this study we tried two other proteases, protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus species. The penicillin-binding protein X (PBP-2X*), a 77-kDa protein, was selected as a model. Like pepsin, neither of these proteases is really specific, but we found very good reproducibility in the digestion pattern. Compared with using pepsin alone, combining the results of the three independent proteolyses increased the coverage for the peptide mapping, thus avoiding missing some potentially interesting regions of the protein. Furthermore, we obtained a better spatial resolution for deuterium incorporation data, specifying accurately the deuterated regions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Notes of a protein crystallographer: the molecular structure of evolutionary theoryACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Cele Abad-Zapatero Some of the current trends in the structure of evolutionary biology are reviewed using as a framework the book of the same title by S. J. Gould (1941,2002). The revised concepts and interpretations of the structure,function relationship in evolutionary biology are discussed in relation to the past achievements and future developments in structural biology. [source] Rotational order,disorder structure of fluorescent protein FP480ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009Sergei Pletnev In the last decade, advances in instrumentation and software development have made crystallography a powerful tool in structural biology. Using this method, structural information can now be acquired from pathological crystals that would have been abandoned in earlier times. In this paper, the order,disorder (OD) structure of fluorescent protein FP480 is discussed. The structure is composed of tetramers with 222 symmetry incorporated into the lattice in two different ways, namely rotated 90° with respect to each other around the crystal c axis, with tetramer axes coincident with crystallographic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates a rotational OD structure with statistically averaged I422 symmetry, although the presence of very weak and diffuse additional reflections suggests that the randomness is only approximate. [source] The Max-Inf2/Lorentz Center workshop on New algorithms in macromolecular crystallography and electron microscopyACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009Navraj S. Pannu The resolution gap between macromolecular crystallography and electron microscopy continues to decrease. Recent advances in specimen preparation, instrumentation and computational power have allowed accurate structure determination of larger macromolecular complexes by crystallography and/or by electron microscopy on cryovitrified samples. New possibilities in structural biology have opened up and new challenges are faced to further reduce the resolution gap. A workshop at the Lorentz Center, Leiden, The Netherlands, which took place in May 2008, was organized to push further the limits of both complementary techniques through improved computational methods. [source] ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignmentsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Charles Simon Bond Marked-up sequence alignments typically provide the central figure in articles describing proteins, whether in the fields of biochemistry, bioinformatics or structural biology. The generation of these figures is often unwieldy: interactive programs are often aesthetically limited and the use of batch programs requires the repetitive iterative editing of scripts. ALINE is a portable interactive graphical sequence-alignment editor implemented in Perl/Tk which produces publication-quality sequence-alignment figures where `what you see is what you get'. ALINE is freely available for download from http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/. [source] First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical valueACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2006L. Banci The EC `Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ,170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples. [source] High-phasing-power lanthanide derivatives: taking advantage of ytterbium and lutetium for optimized anomalous diffraction experiments using synchrotron radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003É. Girard Ytterbium and lutetium are well suited for optimized anomalous diffraction experiments using synchrotron radiation. Therefore, two lanthanide complexes Yb-HPDO3A and Lu-HPDO3A have been produced that are similar to the Gd-HPDO3A complex already known to give good derivative crystals. Derivative crystals of hen egg-white lysozyme were obtained by co-crystallization using 100,mM solutions of each lanthanide complex. De novo phasing has been carried out using single-wavelength anomalous diffraction on data sets collected on each derivative crystal at the LIII absorption edge of the corresponding lanthanide ( = 28,e,). A third data set was collected on a Lu-HPDO3A derivative crystal at the Se,K absorption edge with = 10,e,. The structures were refined and compared with the known structure of the Gd-HPDO3A lysozyme derivative. The quality of the experimental electron-density maps allows easy model building. With LIII absorption edges at shorter wavelengths than the gadolinium absorption edge, lutetium and ytterbium, when chelated by a ligand such as HPDO3A, form lanthanide complexes that are especially interesting for synchrotron-radiation experiments in structural biology. [source] Concanavalin A in a dimeric crystal form: revisiting structural accuracy and molecular flexibilityACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2002Katherine A. Kantardjieff A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56,Å (space group C2221; unit-cell parameters a = 118.70, b = 101.38, c = 111.97,Å; two molecules in the asymmetric unit). The structure has been refined to an Rfree of 0.206 (R = 0.178) after iterative model building and phase-bias removal using Shake&wARP. Correspondence between calculated water,tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided. [source] Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Emerich Mihai Gazdag The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30,mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5,mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research. [source] Glycosaminoglycan in cerebrum, cerebellum and brainstem of young sheep brain with particular reference to compositional and structural variations of chondroitin,dermatan sulfate and hyaluronanBIOMEDICAL CHROMATOGRAPHY, Issue 9 2008Virginia Kilia Abstract Recent advances in the structural biology of chondroitin sulfate chains have suggested important biological functions in the development of the brain. Several studies have demonstrated that the composition of chondroitin sulfate chains changes with aging and normal brain maturation. In this study, we determined the concentration of all glycosaminoglycan types, i.e. chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, hyaluronan and chondroitin in cerebrum, cerebellum and brainstem of young sheep brain. In all cases, chondroitin sulfate was the predominant glycosaminoglycan type, comprising about 54,58% of total glycosaminoglycans, with hyaluronan being present also in significant amounts of about 19,28%. Of particular interest was the increased presence of the disulfated disaccharides and dermatan sulfate in cerebellum and brainstem, respectively, as well as the detectable and measurable occurrence of chondroitin in young sheep brain. Among the three brain areas, cerebrum was found to be significantly richer in chondroitin sulfate and hyaluronan, two major extracellular matrix components. These findings imply that the extracellular matrix of the cerebrum is different from those of cerebellum and brainstem, and probably this fact is related to the particular histological and functional characteristics of each anatomic area of the brain. Copyright © 2008 John Wiley & Sons, Ltd. [source] Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural propertiesBIOTECHNOLOGY JOURNAL, Issue 1 2009Rita Grandori Abstract Since the early 1990s, electrospray-ionization mass spectrometry (ESI-MS) has encountered growing interest as a complementary tool to established biochemical and biophysical methods for investigating protein structure and conformation. Nowadays, applications of ESI-MS to protein investigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, although representing a still very young field. An introductive section presents the major issues related to theoretical and technical aspects of ESI-MS under non-denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained concerning key issues in biotechnology such as stability and aggregation of proteins under both near-native and challenging conditions, and interactions with other proteins, ligands and cofactors. [source] Mechanisms of RNA Degradation by the Eukaryotic ExosomeCHEMBIOCHEM, Issue 7 2010Rafal Tomecki Dr. RNA on its way to destruction: The exosome is a multi-subunit protein complex involved in essentially all phenomena associated with RNA metabolism in eukaryotic cells. This review discusses recent discoveries in the fields of biochemistry and structural biology that have shed new light on the mechanisms of RNA recruitment to the catalytic subunits of the exosome. [source] Supramolecular Domains in Mixed Peptide Self-Assembled Monolayers on Gold NanoparticlesCHEMBIOCHEM, Issue 13 2008Laurence Duchesne Dr. Abstract Self-organization in mixed self-assembled monolayers of small molecules provides a route towards nanoparticles with complex molecular structures. Inspired by structural biology, a strategy based on chemical cross-linking is introduced to probe proximity between functional peptides embedded in a mixed self-assembled monolayer at the surface of a nanoparticle. The physical basis of the proximity measurement is a transition from intramolecular to intermolecular cross-linking as the functional peptides get closer. Experimental investigations of a binary peptide self-assembled monolayer show that this transition happens at an extremely low molar ratio of the functional versus matrix peptide. Molecular dynamics simulations of the peptide self-assembled monolayer are used to calculate the volume explored by the reactive groups. Comparison of the experimental results with a probabilistic model demonstrates that the peptides are not randomly distributed at the surface of the nanoparticle, but rather self-organize into supramolecular domains. [source] | ||||||||||||||||