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A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell-based assays. Solvent-repelling barriers made of Teflon-impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow-through syntheses on paper. The confinement and flow-through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60 % purity for the majority of the peptides (>95 % yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell-based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation. [ABSTRACT FROM AUTHOR]

The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.

As biological macromolecules, proteins are involved in important cellular functions ranging from DNA replication and biosynthesis to metabolic signalling and environmental sensing. Protein sequencing can help understand the relationship between protein function and structure, and provide key information for disease diagnosis and new drug design. Nanopore sensors are a novel technology to achieve the goal of label‐free and high‐throughput protein sequencing. In recent years, nanopore‐based biosensors have been widely used in the detection and analysis of biomolecules such as DNA, RNA, and proteins. At the same time, computer simulations can describe the transport of proteins through nanopores at the atomic level. This paper reviews the applications of nanopore sensors in protein sequencing over the past decade and the solutions to key problems from a computer simulation perspective, with the aim of pointing the way to the future of nanopore protein sequencing. [ABSTRACT FROM AUTHOR]

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [ABSTRACT FROM AUTHOR]

To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail three to five noncovalent interactions important for protein structure and/or function. The assignment is designed to provide a hands-on learning experience for students in a moderately large introductory biochemistry class. [ABSTRACT FROM AUTHOR]

This paper is a further study of ion binding to protein surfaces and builds on the studies of the binding of [Crk(CN)6]3- and [Fe(edta)(H2O)]- previously reported [Williams et al. (1982) FEBS Lett. 15, 293-299; Eley et al. (1982) Eur. J. Biochem. 124, 295-303]. In the present paper the binding of polyaminocarboxylate complexes of gadolinium have been studied. Eight ion-binding sites have been identified on the surface of cytochrome c. These exhibit different binding specificities which, in some cases, are not fully understood. However it is clear that simple outer-sphere interactions are not the sole determining factor for the association of metal ion complexes with proteins. The NMR paramagnetic difference spectrum method has been shown to be good at locating binding sites and revealing qualitative differences in their relative affinities for a range of complex types. However the use of relaxation probes is not a good method for the quantitative determination of binding constants; for this, isostructural shift probes must be sought. [ABSTRACT FROM AUTHOR]

Two papers in this issue of Molecular Microbiology from Carol Gross and colleagues describe experiments that investigate how two alternative Escherichia coliσ proteins recognize target promoter −10 regions. A combination of genetics, biochemistry and bioinformatics is used to show that determinants in two adjacent domains of the σ proteins contact discrete sequence elements in the −10 regions. [ABSTRACT FROM AUTHOR]

This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future. [ABSTRACT FROM AUTHOR]

Not all proteins form well defined three-dimensional structures in their native states. Some amino-acid sequences appear to strongly favour the disordered state, whereas some can apparently transition between disordered and ordered states under the influence of changes in the biological environment, thereby playing an important role in processes such as signalling. Although important biologically, for the structural biologist disordered regions of proteins can be disastrous even preventing successful structure determination. The accurate prediction of disorder is therefore important, not least for directing the design of expression constructs so as to maximize the chances of successful structure determination. Such design criteria have become integral to the construct-design strategies of laboratories within the Structural Proteomics In Europe (SPINE) consortium. This paper assesses the current state of the art in disorder prediction in terms of prediction reliability and considers how best to use these methods to guide construct design. Finally, it presents a brief discussion as to how methods of prediction might be improved in the future. [ABSTRACT FROM AUTHOR]

Identification of high affinity drug‐target interactions is a major research question in drug discovery. Proteins are generally represented by their structures or sequences. However, structures are available only for a small subset of biomolecules and sequence similarity is not always correlated with functional similarity. We propose ChemBoost, a chemical language based approach for affinity prediction using SMILES syntax. We hypothesize that SMILES is a codified language and ligands are documents composed of chemical words. These documents can be used to learn chemical word vectors that represent words in similar contexts with similar vectors. In ChemBoost, the ligands are represented via chemical word embeddings, while the proteins are represented through sequence‐based features and/or chemical words of their ligands. Our aim is to process the patterns in SMILES as a language to predict protein‐ligand affinity, even when we cannot infer the function from the sequence. We used eXtreme Gradient Boosting to predict protein‐ligand affinities in KIBA and BindingDB data sets. ChemBoost was able to predict drug‐target binding affinity as well as or better than state‐of‐the‐art machine learning systems. When powered with ligand‐centric representations, ChemBoost was more robust to the changes in protein sequence similarity and successfully captured the interactions between a protein and a ligand, even if the protein has low sequence similarity to the known targets of the ligand. [ABSTRACT FROM AUTHOR]

Among the variety of transducer concepts proposed for label-free detection of biomolecules, the semiconductor field-effect device (FED) is one of the most attractive platforms. As medical techniques continue to progress towards diagnostic and therapies based on biomarkers, the ability of FEDs for a label-free, fast and real-time detection of multiple pathogenic and physiologically relevant molecules with high specificity and sensitivity offers very promising prospects for their application in point-of-care and personalized medicine for an early diagnosis and treatment of diseases. The presented paper reviews recent advances and current trends in research and development of different FEDs for label-free, direct electrical detection of charged biomolecules by their intrinsic molecular charge. The authors are mainly focusing on the detection of the DNA hybridization event, antibody-antigen affinity reaction as well as clinically relevant biomolecules such as cardiac and cancer biomarkers. [ABSTRACT FROM AUTHOR]

This paper summarises how scaffold proteins affects and regulate the JNK signalling pathway. We believe that some of these scaffold proteins, by virtue of their anchoring and catalytic properties contribute to a high degree of specificity of intra cellular signalling pathways that regulate the progression through the cell cycle. [ABSTRACT FROM AUTHOR]

The potent immunomodulatory, anti-inflammatory and procoagulant properties of protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have previously been found to be modulated by a supramolecular monomer–trimer equilibrium. More structural details that integrate experimental data into a predictive framework have recently been reported. Unfortunately, homology modelling and fold-recognition strategies were not successful in creating a theoretical model of the structural organization of SV-IV. It was inferred that the global structure of SV-IV is not similar to that of any protein of known three-dimensional structure. Reversing the classical approach to the sequence–structure–function paradigm, in this paper we report novel information obtained by comparing the physicochemical parameters of SV-IV with two datasets composed of intrinsically unfolded and ideally globular proteins. In addition, we analyse the SV-IV sequence by several publicly available disorder-oriented predictors. Overall, disorder predictions and a re-examination of existing experimental data strongly suggest that SV-IV needs large plasticity to efficiently interact with the different targets that characterize its multifaceted biological function, and should therefore be better classified as an intrinsically disordered protein. [ABSTRACT FROM AUTHOR]

Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007) Eur. J. Neurosci., 26, 547–559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (∼1 m) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 whileL-intersectin, H- andL-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release. [ABSTRACT FROM AUTHOR]

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-α-helix bundle with an up-down-up topology and a 310-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters. [ABSTRACT FROM AUTHOR]

Proteomic tools—in particular, mass spectrometry (MS)—have advanced significantly in recent years, and the identification of proteins within complex mixtures is now a routine procedure. Quantitative methods of analysis are less well advanced and continue to develop. These include the use of stable isotope ratio approaches, isotopically labeled peptide standards, and nonlabeling methods. This paper summarizes the use of MS as a proteomics tool to identify and semiquantify proteins and their modified forms by using examples of relevance to the Maillard reaction. Finally, some challenges for the future are presented. [ABSTRACT FROM AUTHOR]

The article present abstracts of various papers that were presented during the Scandinavian Physiological Society meeting titled "Transport and Membrane Trafficking in Cells" that was held form May 7-9, 2004 at Goteborg University, Lundsbrunn Spa. One of the papers discussed is "Trafficking of Ion Transport Proteins: New Partners and Potential Therapies," by M.J. Caplan, A. Duffield, E. Kamsteeg, J. Gloeckner-Pagel and M.E. Egan. The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The authors are interested in the molecular signals and interactions that control the sorting and regulation of ion transport proteins in polarized cells. Another paper discussed is "Receptor Dynamics at Synapses: A View From Single Molecule Imaging," by A. Triller.

The ability ofEntamoeba histolyticaTATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study thein vitroEhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box forE. histolytica, was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in supershift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found inE. histolyticagene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (±0.39) × 10−11 and 1.60 (±0.37) × 10−10 m. TATTTAAA and TAT_ _AAA motifs presented the lowestKD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding. [ABSTRACT FROM AUTHOR]

The DELLA proteins are a family of putative transcriptional regulators that inhibit the cell proliferation and expansion that drives the growth of plant organs. Plant growth is stimulated via destruction of DELLA proteins in the 26S proteasome. Recent advances have shown that the‘classical’ phytohormones gibberellin (GA), auxin and ethylene control plant growth by influencing DELLA-destruction. This paper outlines the discovery, molecular characterization and biology of the DELLA proteins, and summarizes the evidence that they act as integrators of response to multiple growth-regulatory signals. [ABSTRACT FROM AUTHOR]

Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein–protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels. [ABSTRACT FROM AUTHOR]

In this paper, we studied the fate of endocytosed glycosyiphosphatidyl inositol anchored proteins (GPI-APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway. [ABSTRACT FROM AUTHOR]

Probabilistic direct-methods phasing theory, origin ally based on a uniform atomic distribution hypothesis, is shown to be adaptable to a non-uniform bulk-solvent-compensated globbic approximation for protein crystals at low resolution. The effective number ng of non-H protein atoms per polyatomic glob increases with decreasing resolution; low-resolution phases depend on the positions of only Ng = Na/Ng globs rather than Na atoms. Test calculations were performed with measured structure-factor data and the refined structural parameters from a protein crystal with ∼ 10 000 non-H protein atoms per molecule and ∼ 60% solvent volume. Low-resolution data sets with dmin ranging from 15 to 5 Å gave ng = admin + b, with a = 1.0 Å-1 and b = -1.9 for the test case. Results of tangent- formula phase-estimation trials emphasize that completeness of the low-resolution data is critically important for probabilistic phasing. [ABSTRACT FROM AUTHOR]

Confinement of reactive chemical species uniquely affects chemical reactivity by restricting the physical space available and by restricting access to interactions with the solvent. In Nature, for example, confined protein binding pockets govern processes following photoisomerization reactions and the isomerizations themselves. Here we describe the first example of a dihydroazulene/vinylheptafulvene (DHA/VHF) photo-switch functioning in water, and we show how its switching behavior is strongly influenced by supramolecular interactions with a series of cucurbit[ n]uril (CB) host molecules. In CB7 inclusion complexes, the kinetics of the thermal VHF-to-DHA back-reaction is accelerated, while in CB8 inclusion complexes, the kinetics is slowed down as compared to the free photo-switch. The effect of the CB encapsulation of the photo-switch can be effectively canceled by introducing a guest that binds the CB more strongly. According to DFT calculations, a stabilization of the reactive s- cis VHF conformer relative to the s- trans VHF appears to be a contributing factor responsible for the accelerated back-reaction when encapsulated in CB7. [ABSTRACT FROM AUTHOR]

The activities of five enzymes were determined by flurometric micromethods on epidermal tumors (basal and squamous cell epithelioma, keratoacanthoma and verruca seborreica) and compared to the activities in the adjoining, apparently normal epidermis. All activities were higher in the tumoral tissues. [ABSTRACT FROM AUTHOR]

Epizootic haematopoietic necrosis virus (EHNV) has been isolated from redfin perch, Perca fluviatilis L., and cultured rainbow trout, Oncorhynchus mykiss (Walbaum), in Victoria and New South Wales, Australia. On the basis of virion size, icosahedral morphology and ultrastructural characteristics of virus-infected cells, EHNV was designated as an iridovirus. The following observations outlined in this paper confirm the designation: (1) The number and complexity of proteins in purified EFINV and the ordered appearance of virus-specific proteins in infected cells are similar to that described for the iridovirus, frog virus 3 (FV3). (2) EHNV isolated from the cytoplasm of infected cells contains a lipid membrane associated with 7-9 nm capsomeric subunits containing a 52-ID protein. (3) The morphogenesis of EHNV within cytoplasmic inclusion bodies resembles that of FV3 and a piscine iridovirus isolated from goldfish, Carassius auratus (L.) (4) EHNV also resembles FV3 in its association with the cytoskeleton of infected cells. (5) EHNV DNA, like that of other iridoviruses, is resistant to digestion with Hpall, a restriction endonuclease which does not cleave DNA at CCGG sequences where the internal cytosine is methylated. [ABSTRACT FROM AUTHOR]

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 °C, echistatin contains no α-helix but contains mostly β-turns and β-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 °C and 72 °C being irreversible. Raman spectra also indicate considerable β-turn and β-sheet (20%) structure and an absence of α-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 °C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either α-helical or β-sheet conformation. A number of turns could be characterised. An irregular β-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies. [ABSTRACT FROM AUTHOR]

Shigella flexneri, a Gram-negative bacillus belonging to the family Enterobacteriaceae, causes bacillary dysentery in humans by invading colonic epithelial cells. Processes by which epithelial cells, which are not professional phagocytes, may limit the spread of the invading microorganisms are poorly understood. This paper shows that IcsA (VirG), a 120kDa bacterial outer membrane protein responsible for intracellular and cell-to-cell spread through polymerization of actin, is a major substrate for phosphorylation by cyclic-dependent protein kinases. Site-directed mutagenesis of a sequence encoding phosphorylation consensus motif SSRRASS, located at residues 754-760, almost completely abolished the ability of this protein to be phosphorylated by protein kinase A. Such mutants expressed a 'super lcs' phenotype, characterized by an increased capacity to spread from cell-to-cell during the first three hours of infection in the HeLa cell infection assay. These data suggest that host-cell phosphorylation of key virulence proteins located on the bacterial surface may represent a significant host defence mechanism during the invasion process. [ABSTRACT FROM AUTHOR]

The complete primary structure of the constant part of the μ-chain-disease protein, BOT, was established. It includes the whole CH2, CH3 and CH4 domains. Two amino acid changes were found, at positions 309 (Ser ↵ Gly) and 333 (Val ↵ Gly) (GAL numbering). In two additional monoclonal n chains (SCO and CO), the same positions showed an amino acid variability. From these data it may be concluded that four types of μ chains exist in the human: (1) GAL type with Ser-309 and Val-333; (2) OU type with Gly-309 and Val-333; (3) SCO type with Ser-309 and Gly-333; (4) BOT/CO type with Gly-309 and Gly-333. The meaning of this molecular polymorphism is discussed. [ABSTRACT FROM AUTHOR]

In this paper the mode of action of IF-1 in 40-S initiation complex formation was studied with MS 2 RNA as messenger. Using initiation factors IF-2 and IF-3 labeled in vitro it appeared that IF-I did not influence the binding of these factors in the absence of fMet-tRNA. However, in the presence of fMet-tRNA it was found that the enhancement of the fMet-tRNA binding by IF-1 was accompanied with an equimolar increase in binding of IF-2. Moreover, it appeared that also in absence of IF-I, fMet-tRNA binding is coupled with an equimolar enhancement of the IF-2 binding, which suggests the existence of a preribosomal complex between IF-2 and fMet-tRNA. The apparent K,, values for both the binding of fMet-tRNA and IF-2 to 30-S subunits were determined and appeared to be equal, which makes a functioning of such a preribosomal complex in protein initiation very likely. The participation of GTP in this complex will be discussed. Functions of IF-I in dissociation and recycling of IF-2, described by others, and the stimulation on the 30-S subunit level might well be explained as pleiotropic effects of one basic action of IF-1, i.e. a conformational change of 30-S subunits. [ABSTRACT FROM AUTHOR]

In the accompanying paper, we present evidence to show that the biosynthesis of the two characteristic polypeptide constituents ‘A’ and ‘B’ of the crystal protein toxin of Bacillus thuringiensis car. tolworth, is restricted to a short interval, from 16–18 h, in the mid-stationary phase of the growth cycle of 24 h in culture. Here we examine the question of the kinetics of biosynthesis of the corresponding crystal protein messenger RNA. We have taken the direct, approach of assaying a series of RNAs isolated from B. thuringiensis cultures at varying times of culture for messenger activity in a heterologous Escherichia coli, subcelular system. The product of RNA-stimulated protein synthesis in vitro was characterized by immunoprecipitation with anti-serum against purified crystal protein and by polyacrylamide-gel electrophoresis of the antigen-antibody precipitates dissolved in the detergent sodium dodecylsulfate. RNA from B. thuringiensis, but not that of Bacillus cerens, stimulates the synthesis of protein which specifically precipitates with crystal protein antiserum. The time of extraction of RNA from B. thurinrjgiensis does not appear to be critical in determining its messenger activity : all samples taken in the interval of 11–20 h contained messenger RNA for crystal protein by the criterion of specific immunoprecipitation. In each case a significant proportion of the synthesized protein migrated in the region of the smaller-molecular-weight component A (55000) of the crystal protein in the electrophoretic separation. However, hardly any synthesized protein co-migrated with the larger-molecular-weight component B (120000) of this protein. Since all the labelled protein precipitated by the crystal protein antiserum was larger than about 15000 molecular weight we conclude that mainly large fragments of the crystal protein were synthesized in the E. coli system. We failed in our attempts to isolate polysomes as a source of messenger RNA from stationary-phase ceils. Thus it was not possible to determine by experiments similar to those described above whether the crystal protein messenge is stored in ribosomes or in some other fraction of the cell when it is not being translated. [ABSTRACT FROM AUTHOR]

In 2012, preliminary guidelines were published addressing sample quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular small-angle scattering (SAS) experiments. Biomolecular SAS has since continued to grow and authors have increasingly adopted the preliminary guidelines. In parallel, integrative/hybrid determination of biomolecular structures is a rapidly growing field that is expanding the scope of structural biology. For SAS to contribute maximally to this field, it is essential to ensure open access to the information required for evaluation of the quality of SAS samples and data, as well as the validity of SAS-based structural models. To this end, the preliminary guidelines for data presentation in a publication are reviewed and updated, and the deposition of data and associated models in a public archive is recommended. These guidelines and recommendations have been prepared in consultation with the members of the International Union of Crystallography (IUCr) Small-Angle Scattering and Journals Commissions, the Worldwide Protein Data Bank (wwPDB) Small-Angle Scattering Validation Task Force and additional experts in the field. [ABSTRACT FROM AUTHOR]

This paper reports a non-destructive method for evaluating hair interior and surface damage based on near-infrared (NIR) diffuse reflectance (DR) spectroscopy. It is important to know the extent of chemical damage in the interior and surface proteins of the hair in order to choose an appropriate restoration agent or chemical treatment. Unfortunately, though there are many simple and non-destructive methods for evaluating the hair surface, the existing evaluation methods for monitoring chemical changes in the interior proteins are very complicated and destructive. Therefore, we have attempted to develop a new non-destructive method to evaluate the damage of the hair interior and surface simultaneously by using NIR-DR spectroscopy. The key to this study was the combined application of NIR-DR spectroscopy and principal component analysis to development of a method for the evaluation of hair damage and finding the most suitable wavenumber region (5060—4500 cm-1) for this evaluation. In this study, we developed a new evaluation method that can indicate hair interior and surface damage conditions induced by chemical treatments in a simple, rapid, non-destructive manner based on NIR-DR using a fibre probe on hair Paper presented at the IFSCC International Conference 2005, Florence, Italy. [ABSTRACT FROM AUTHOR]

The interfacial properties of solid substrates are of importance for protein adsorption. Herein, we report a reversible protein adsorption switch based on the host-guest interaction of the butoxy pillar[5]arene and adipic acid. By the detector of the contact angle (CA), atomic force microscopy (AFM), and luminoscope on the silicon substrate, the intelligent protein switch exhibits excellent adsorptivity for BSA and switch performance by pH regulation. [ABSTRACT FROM AUTHOR]

The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for Nα-deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca2+ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production. [ABSTRACT FROM AUTHOR]

Synthetic biology is first represented in terms of two complementary aspects, the bio-engineering one, based on the genetic manipulation of extant microbial forms in order to obtain forms of life which do not exist in nature; and the chemical synthetic biology, an approach mostly based on chemical manipulation for the laboratory synthesis of biological structures that do not exist in nature. The paper is mostly devoted to shortly review chemical synthetic biology projects currently carried out in our laboratory. In particular, we describe: the minimal cell project, then the “Never Born Proteins” and lastly the Never Born RNAs. We describe and critically analyze the main results, emphasizing the possible relevance of chemical synthetic biology for the progress in basic science and biotechnology. [ABSTRACT FROM AUTHOR]

The sequence of the Streptococcus equi subspecies equi (S equi) M-like protein (SeM) gene was determined for 105 isolates of S equi from strangles outbreaks in the UK during 2010 and compared with previous data from 2007 to 2008. Twenty-three distinct alleles were identified, including 11 novel alleles. One allele giving rise to a putative truncated M protein was identified from the guttural pouch of an asymptomatic carrier. Allele 9 was the most prevalent, comprising 57.7 per cent of isolates, followed by allele 6 (10.3 per cent). Significant changes in allele prevalence were found between 2007, 2008 and 2010, with an increasing prevalence in SeM-9-related alleles and a corresponding decreasing prevalence in SeM-6-related alleles observed over the period (P<0.001). Geographical proximity of outbreaks caused by some uncommon alleles was apparent between 2007, 2008 and 2010. [ABSTRACT FROM AUTHOR]

The structure and stability of repeat proteins has been little studied in comparison to the properties of the more familiar globular proteins. Here, the structure and stability of designed tetratricopeptide-repeat (TPR) proteins is described. The TPR is a 34-amino-acid motif which adopts a helix–turn–helix structure and occurs as tandem repeats. The design of a consensus TPR motif (CTPR) has previously been described. Here, the crystal structures and stabilities of proteins that contain eight or 20 identical tandem repeats of the CTPR motif (CTPR8 and CTPR20) are presented. Both CTPR8 and CTPR20 adopt a superhelical overall structure. The structures of the different-length CTPR proteins are compared with each other and with the structures of natural TPR domains. Also, the unusual and perhaps unique crystal-packing interactions resulting in pseudo-infinite crystalline superhelices observed in the different crystal forms of CTPR8 and CTPR20 are discussed. Finally, it is shown that the thermodynamic behavior of CTPR8 and CTPR20 can be predicted from the behavior of other TPRs in this series using an Ising model-based analysis. The designed protein series CTPR2–CTPR20 covers the natural size repertoire of TPR domains and as such is an excellent model system for natural TPR proteins. [ABSTRACT FROM AUTHOR]

The crystal structures of homodimeric thioredoxin reductase (TrxR) from the gastric pathogen Helicobacter pylori in complex with NADP+ have been determined for the oxidized and reduced form of the enzyme at 1.7 and 1.45 Å resolution, respectively. The enzyme subunit is built up of FAD- and NAD(P)H-binding domains, each of which contain variants of the Rossmann fold typical of nucleotide-binding proteins. The majority of the amino-acid residues binding the cofactors FAD and NAD(P)H are conserved in the low-molecular-weight thioredoxin reductases. In the reduced species the isoalloxazine ring of FAD is bent along an axis passing through the N5 and N10 atoms with an angle of 22° and the ribityl moiety adopts an unusual conformation. Well defined electron density shows the position of the whole NADP+ molecule with a binding mode similar to that observed in the Escherichia coli TrxR–thioredoxin complex, although the orientation of the NAD(P)H-binding domain is different. Rigid-body rotation of this domain to the orientation observed in the E. coli TrxR–thioredoxin complex positions NADP+ adjacent to the FAD molecule, suitable for electron transfer, without any readjustments of the conformation of NADP+. A comparison of the binding surfaces of thioredoxin and thioredoxin reductases from H. pylori and E. coli shows that the overall surface charge distribution in these proteins is conserved and that residue substitutions that change the shape of the binding surface may account for the species-specific recognition of thioredoxin by TrxR. [ABSTRACT FROM AUTHOR]

A Bence Jones protein isolated in the early 1960s from a patient (initials KWR) suffering from plasma-cell dyscrasia was crystallized and its structure was analyzed in four different unit cells by X-ray diffraction. The final models of the molecule in all crystal forms were virtually the same, although the elbow angles relating the constant and variable domains of the Bence Jones dimers varied over a range of 10°. The tetragonal form had an R factor of 22.6% and an Rfree of 28.3% at 2.2 Å resolution. Phosphate or sulfate ions (depending on the crystallization conditions) were found in the antigen-combining sites in all crystals, as well as an unidentified ligand tightly bound in the hydrophobic `deep pocket' beneath the antigen-binding site. The ligand was treated as a phenol molecule. Two trigonal crystal forms were among those solved. One was grown at pH 4.0 and the other was only obtained after sitting for more than eight months at room temperature. The latter crystal was composed of molecules that were degraded in their constant domains. Both low pH and proteolytic degradation of constant domains are known to promote the polymerization of some Bence Jones proteins into amyloid fibrils. Indeed, in both trigonal crystal forms the molecules are organized with pseudo-hexagonal symmetry about the unique crystallographic axes in a manner suggestive of such fibrils. The arrangement of Bence Jones dimers is also consistent with other observations regarding Bence Jones amyloid-fibril structure and current models. [ABSTRACT FROM AUTHOR]

The Protein Data Bank and Cambridge Structural Database were analyzed with the aim of verifying whether the restraints that are most commonly used for protein structure refinement are still appropriate 15 years after their introduction. From an analysis of selected main-chain parameters in well ordered fragments of ten highest resolution protein structures, it was concluded that some of the currently used geometrical target values should be adjusted somewhat (the C—N bond and the N—Cα—C angle) or applied with less emphasis (peptide planarity). It was also found that the weighting of stereochemical information in medium-resolution refinements is often overemphasized at the cost of the experimental information in the diffraction data. A correctly set balance will be reflected in root-mean-square deviations from ideal bond lengths in the range 0.015–0.020 Å for structures refined to R factors of 0.15–0.20. At ultrahigh resolution, however, the diffraction terms should be allowed to dominate, with even higher acceptable deviations from idealized standards in the well defined fragments of the protein. It is postulated that modern refinement programs should accommodate variable restraint weights that are dependent on the occupancies and B factors of the atoms involved. [ABSTRACT FROM AUTHOR]

The practical applicability of in-house structure determination using Cr Kα X-rays (2.29 Å) and a loopless free crystal-mounting method was examined using five novel proteins. Proteins from 9.6 to 84 kDa have been solved using this method without any derivatization. In all cases, more than 90% of structures were constructed automatically with side chains by use of the Cr SAD method. The free crystal-mounting technique increases the accuracy of the anomalous differences between Bijvoet mates and makes the in-house single-wavelength SAD method with a Cr Kα X-ray source a very useful tool for high-throughput structure determination. In addition, a Cr/Cu dual-wavelength system makes it possible to perform structure analysis from phasing to refinement of the structure in-house. [ABSTRACT FROM AUTHOR]

The yeast proteins Dsk2 and Rad23 belong to a family of proteins that contain an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). Both Dsk2 and Rad23 function as adaptors to target ubiquitin- labelled proteins to the proteasome through recognition of polyubiquitin (four or more K48-linked ubiquitins) by their UBA domains and to the yeast proteasomal subunit Rpn1 by their UBL domains. The crystal structures of the Dsk2 UBL domain, the Dsk2 UBA domain and the Dsk2 UBA-UBL complex are reported. In the crystal, the Dsk2 UBA domains associate through electrostatic interactions to form ninefold helical ribbons that leave the ubiquitin-binding surface exposed. The UBA-UBL complex explains the reduced affinity of the UBA domain for UBL compared with ubiquitin and has implications for the regulation of Dsk2 adaptor function during ubiquitin -mediated proteasomal targeting. A model is discussed in which two or more Dsk2 UBA molecules may selectively bind to K48-linked polyubiquitin. [ABSTRACT FROM AUTHOR]

ACORN has previously been shown to provide an efficient density-modification procedure for the solution of protein structures using diffraction data to better than 1.3 Å. The initial phase set could be obtained from a variety of sources such as the position of a heavy atom, a set of scatterers such as S that had been positioned from anomalous dispersion measurements, a fragment or a very low homology model placed from a molecular-replacement search. Several structures solved using the early version of ACORN have been reported in the literature. Here, the effect of applying the original ACORN procedures at lower resolution is reported and new procedures that yield good-quality maps with data sets of resolution down to 1.7 Å are described. These new procedures involve the artificial extension of data to atomic resolution and new density-modification processes that develop density at atomic positions that was previously suppressed. The test calculations were aimed firstly towards a proof of principle using a small fragment of a known structure to demonstrate that the procedure could generate correct density and a derived model in initially empty regions of the cell. Further tests addressed the use of more realistic starting models. [ABSTRACT FROM AUTHOR]

A Monte Carlo procedure is described that generates random structure factors with simulated errors corresponding to an X-­ray data set of a protein of a specific size and given heavy-atom content. The simulated data set can be used to estimate Bijvoet ratios and figures of merit as obtained from SAD phasing routines and can be used to gauge the feasibility of solving a structure via the SAD method. In addition to being able to estimate results from phasing, the simulation allows the estimation of the correlation coefficient between |Δ F|, the absolute Bijvoet amplitude difference, and FA, the structure-factor amplitude of the heavy-atom model. As this quantity is used in various substructure-solution routines, the estimate provides a rough estimate of the ease of substructure solution. Furthermore, the Monte Carlo procedure provides an easy way of estimating the number of significant Bijvoet intensity differences, denoted as the measurability, and is proposed as an intuitive measure of the quality of anomalous data. [ABSTRACT FROM AUTHOR]

Abstract: Most protein structure prediction methods use templates to assist in the construction of protein models. In this paper, we analyse the current state of template-based modelling approaches and reach an estimate of the empirical limits of these methods. Our analysis show that current prediction methods are already reaching these empirical accuracy limits in the easier cases, where finding a close homologue to the native target structure is not a problem. However, we find that even in the absence of alignment errors and using optimal templates, template-based methods have intrinsic limitations, suggesting that other methodologies, such as ab initio procedures, must be used if accuracy is ultimately to be improved. [Copyright &y& Elsevier]

The structure of the Hfq protein from Pseudomonas aeruginosa was determined using two different ionic conditions. In both cases the molecules formed identical hexameric rings, but some variations in the crystal packing were revealed. Hfq belongs to the family of Sm/LSm proteins, the members of which can form hexameric as well as heptameric rings. Comparative analysis of known structures of this protein family shows that the fragment of the Sm-fold responsible for oligomerization is strongly structurally conserved. In the heptameric ring, three conserved hydrogen bonds between β-strands of adjacent molecules hold together the monomers, whereas in the hexameric rings of Hfq an additional conserved inaccessible hydrogen bond between neighbouring monomers is observed. [ABSTRACT FROM AUTHOR]

The article focuses on the variation of the values of dihedral angles in proteins. The variation is divided into two categories by analyzing distributions in a database of structures determined at a resolution of 1.8 Å or better. Each dihedral angle of any given side chain in a protein has its own specific equilibrium value which is determined by the details of the packing of the rest of the protein around the side chain. In order to determine the distribution of side-chain dihedral angles X-ray crystallography is beset by errors in the experimental. Thermal motion, static disorder and irregularities of the crystal lattice further increase the width of the distribution.

Crystal structures are reported for free and coenzyme. A (CoA) bound forms of the YfdW protein from Escherichia coli, a representative type III CoA transferase. The structures reveal a two-domain protomer with interdomain connections forming a ring-like structure with a large central hole. Two protomers associate to form a highly intertwined dimer in which the hole of each ring is filled by the partner molecule. Each protomer binds a single CoA molecule and these CoA-binding sites are distant from one another in the dimer. [ABSTRACT FROM AUTHOR]

Describes the preparation and crystallization of five different metal-substituted forms of Clostridium pasteurianum rubredoxin. Isomorphism of all five forms of rubredoxin crystallized; Data indicating that a knowledge of these structures contributes to a molecular understanding of the function of the iron-sulfur electron-transport protein.

Details the computation of the profiles of squared normalized structure factors for a large number of proteins and nucleic acids. Data showing that the solvent dip at around 6.3 A resolution is to a large extent a protein secondary-structure effect that is enhanced by the water structure.