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Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation- contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR21-606 N-terminal fragment. Soluble hRyR21-606 was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95 purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.

ABSTRACTThe trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. bruceihas been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies—troublesome proteins and technical limitations—are common not only to T. bruceibut also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.

We review the most recent findings on human filamin structure, with particular emphasis on the relationships between structure, function, and interaction. Filamin is a cytoskeletal actin-binding protein and it is therefore crucial in providing cells with the necessary mechanical and dynamical properties. Filamentous actin cross-linking by filamin is regulated by a number of other proteins and the molecular mechanisms of this complex interaction network can be understood by highlighting the structural features of isolated filamin moieties and of their complexes with several partners. Here we describe first the structure-function relationships of the isolated filamin, its flexibility, and its dimerization mechanism. Secondly, we illustrate the structural mechanism with which filamin can recognize its partners, both the actin filaments and the regulatory proteins.

N2O is generated by denitrifying bacteria as a product of NO reduction. In denitrification, N2O is metabolized further by the enzyme N2O reductase (N2OR), a multicopper protein which converts N2O into dinitrogen and water. The structure of N2OR remained unknown until the recent elucidation of the structure of the enzyme isolated from Pseudomonas nautica. In the present paper, we report the crystal structure of a blue form of the enzyme that was purified under aerobic conditions from Paracoccus denitrificans. N2OR is a head-to-tail homodimer stabilized by a multitude of interactions including two calcium sites located at the intermonomeric surface. Each monomer is composed of two domains: a C-terminal cupredoxin domain that carries the dinuclear electron entry site known as CuA, and an N-terminal seven-bladed β-propeller domain which hosts the active-site centre CuZ. The electrons are transferred from CuA to CuZ across the subunit interface. CuZ is a tetranuclear copper cluster in which the four copper ions (Cu1 to Cu4) are ligated by seven histidine imidazoles, a hydroxyl or water oxygen and a bridging inorganic sulphide. A bound chloride ion near the CuZ active site shares one of the ligand imidazoles of Cu1. This arrangement probably influences the redox potential of Cu1 so that this copper is stabilized in the cupric state. The treatment of N2OR with H2O2 or cyanide causes the disappearance of the optical band at 640nm, attributed to the CuZ centre. The crystal structure of the enzyme soaked with H2O2 or cyanide suggests that an average of one copper of the CuZ cluster has been lost. The lowest occupancy is observed for Cu3 and Cu4. A docking experiment suggests that N2O binds between Cu1 and Cu4 so that the oxygen of N2O replaces the oxygen ligand of Cu4. Certain ligand imidazoles of Cu1 and Cu2, as well as of Cu4, are located at the dimer interface. Particularly those of Cu2 and Cu4 are parts of a bonding network which couples these coppers to the CuA centre in the neighbouring monomer. This structure may provide an efficient electron transfer path for reduction of the bound N2O.

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N2O to N2. The crystal structure of N2ORs from Pseudomonas nautica(Pn) and Paracoccus denitrificans(Pd) were solved at resolutions of 2.4 and 1.6 Å, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol.7, 191–195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.

Prokaryotic Cu,Zn superoxide dismutases are characterized by a distinct quaternary structure, as compared to that of the homologous eukaryotic enzymes. Here we report a newly determined crystal structure of the dimeric Cu,Zn superoxide dismutase from Photobacterium leiognathi(crystallized in space group R32, refined at 2.5 Å resolution, R-factor 0.19) and analyse it in comparison with that of the monomeric enzyme from Escherichia coli. The dimeric assembly, observed also in a previously studied monoclinic crystal form of P. leiognathiCu,Zn superoxide dismutase, is based on a ring-shaped subunit contact region, defining a solvated interface cavity. Three clusters of neighbouring residues play a direct role in the stabilization of the quaternary assembly. The present analysis, extended to the amino acid sequences of the other 11 known prokaryotic Cu,Zn superoxide dismutases, shows that at least in five other prokaryotic enzymes the interface residue clusters are under strong evolutionary constraint, suggesting the attainment of a quaternary structure coincident with that of P. leiognathiCu,Zn superoxide dismutase. Calculation of electrostatic fields for both the enzymes from E. coliand P. leiognathishows that the monomeric/dimeric association behaviour displayed by prokaryotic Cu,Zn superoxide dismutases is related to the distribution of surface charged residues. Moreover, Brownian dynamics simulations reproduce closely the observed enzyme:substrate association rates, highlighting the role of the active site neighbouring residues in determining the dismutase catalytic properties.

A simple pressure cell is described that may be used to bind xenon to protein crystals freely suspended in thin films which may then be flash‐cooled, thus immobilizing the bound xenon. As an example, the use of the cell in the structure determination of perchloric‐acid‐soluble protein, by the multiple isomorphous replacement method, is described.

This issue of Current Protein and Peptide Science is devoted to the emerging field of likelihood of protein crystallization and is related to the seminars and lectures presented recently at the Workshop on the definition of protein domains and their likelihood of crystallization, held in Vienna at the end of June 2006 (http://www.emblhamburg. de/workshops/2006/domains/), where a number of scientists addressed these questions by presenting and debating both experimental and computational approaches. Likelihood of crystallization must be predicted computationally and/or determined experimentally in order to avoid time expensive experiments on samples, the three-dimensional structure of which cannot be determined experimentally, because of a series of possible obstacles. For example, if a protein is natively disordered, in the sense that it is not characterized by a unique, well defined conformation, its three-dimensional structure cannot be determined experimentally, since it does not exist. Moreover, a sequence construct that does not correspond to a protein domain might be difficult to express because of its misfolding or its reduced solubility. This is particularly important in the structural genomics era, in which high throughput approaches are applied to the determination of three-dimensional structures of proteins, the biochemical, biophysical, and biological features of which were not previously studied. However, the preliminary analysis and estimation of the likelihood of crystallization is not relegated to proteomics studies only, but it is important also for traditional hypothesis driven projects, in which the optimization of the protein sample is equally important, allowing one to generate samples suitable for structural studies and/or improve diffraction quality of crystals and obtain, as a consequence, more reliable final results. The first review, written by Dmitrij Frishman and co-workers (Technische Universitat Munchen, Germany), deals with the general problem of predicting, with computational and bioinformatics methods, experimental success in cloning, expression, soluble expression, purification and crystallization of proteins. On the basis of publicly available resources, sophisticated machine learning algorithms allow one to make reasonable predictions. For example, solubility predictions are reaching the accuracy of over 70%. The successive four reviews are devoted to prediction, determination, and analysis of conformational disorder. Sonia Longhi and co-workers (CNRS and Universites Aix-Marseille I et II, France) presents an overview of several methods currently employed for predicting protein conformational disorder and present some practical examples of how they can be combined in order to achieve more reliable predictions. Anne Poupon and co-workers (Universite Paris-Sud, France) report the high throughput application of disorder predictions in a structural genomics project on soluble yeast proteins and focus their attention on strategies for tailoring proteins into crystallizable domains. Predictions of conformational disorder are analyzed also by Zsuzsanna Dosztanyi and co-workers (Hungarian Academy of Sciences, Hungary), though from a different perspective. The primary focus of this review is the systematic interpretation of the scores of different predictors. Experimental approaches for the detection of protein disorder are reviewed by Peter Tompa and co-workers (Hungarian Academy of Sciences, Hungary), with special emphasis on proteomic-scale methods, like heat- or acid treatments with a subsequent two-dimensional electrophoresis/mass spectrometry characterization. Furthermore, the problem of defining domain boundaries on the basis of the amino acidic sequences is analyzed in the next two reviews. David Jones and co-workers (University College London, United Kingdom) compare completely automatic and computer-assisted methods and discuss the problem of benchmarking different predictors. Furthermore, the DomPred server, which includes predictors based on sequence comparisons and on secondary structure predictors, is critically analyzed in order to allow its optimal use.......