Integrated structure-based protein interface prediction.

BACKGROUND: Identifying protein interfaces can inform how proteins interact with their binding partners, uncover the regulatory mechanisms that control biological functions and guide the development of novel therapeutic agents. A variety of computational approaches have been developed for predicting a protein's interfacial residues from its known sequence and structure. Methods using the known three-dimensional structures of proteins can be template-based or template-free. Template-based methods have limited success in predicting interfaces when homologues with known complex structures are not available to use as templates. The prediction performance of template-free methods that only rely only upon proteins' intrinsic properties is limited by the amount of biologically relevant features that can be included in an interface prediction model. RESULTS: We describe the development of an integrated method for protein interface prediction (ISPIP) to explore the hypothesis that the efficacy of a computational prediction method of protein binding sites can be enhanced by using a combination of methods that rely on orthogonal structure-based properties of a query protein, combining and balancing both template-free and template-based features. ISPIP is a method that integrates these approaches through simple linear or logistic regression models and more complex decision tree models. On a diverse test set of 156 query proteins, ISPIP outperforms each of its individual classifiers in identifying protein binding interfaces. CONCLUSIONS: The integrated method captures the best performance of individual classifiers and delivers an improved interface prediction. The method is robust and performs well even when one of the individual classifiers performs poorly on a particular query protein. This work demonstrates that integrating orthogonal methods that depend on different structural properties of proteins performs better at interface prediction than any individual classifier alone.

Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor.

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

EVA: continuous automatic evaluation of protein structure prediction servers.

UNLABELLED: Evaluation of protein structure prediction methods is difficult and time-consuming. Here, we describe EVA, a web server for assessing protein structure prediction methods, in an automated, continuous and large-scale fashion. Currently, EVA evaluates the performance of a variety of prediction methods available through the internet. Every week, the sequences of the latest experimentally determined protein structures are sent to prediction servers, results are collected, performance is evaluated, and a summary is published on the web. EVA has so far collected data for more than 3000 protein chains. These results may provide valuable insight to both developers and users of prediction methods. AVAILABILITY: http://cubic.bioc.columbia.edu/eva. CONTACT: eva@cubic.bioc.columbia.edu

Predicting protein conformation by statistical methods.

The unique folded structure makes a polypeptide a functional protein. The number of known sequences is about a hundred times larger than the number of known structures and the gap is increasing rapidly. The primary goal of all structure prediction methods is to obtain structure-related information on proteins, whose structures have not been determined experimentally. Besides this goal, the development of accurate prediction methods helps to reveal principles of protein folding. Here we present a brief survey of protein structure predictions based on statistical analyses of known sequence and structure data. We discuss the background of these methods and attempt to elucidate principles, which govern structure formation of soluble and membrane proteins.

Altered subunit communication in subfamilies of trimeric dUTPases.

The enzyme dUTPase is essential in preventing uracil incorporation into DNA. Design of antagonists against this novel chemotherapeutic target requires identification of species-specific differences in the structure and mechanism of the enzyme. This task is now approached via comparisons of available crystallographic structures of dUTPases from Homo sapiens, Escherichia coli, and retroviruses. The eukaryotic protein uniquely displays polar and charged amino acid residues participating in threefold intersubunit interactions. In bacterial and retroviral dUTPases, threefold interactions are mainly hydrophobic. The residues responsible for this contrast are mapped in multiple sequence alignment to positions differently and characteristically conserved in distinct evolutionary branches. The general feature of this contrast is further strengthened by a second eukaryotic model structure constructed using comparative modeling. The dUTPase cDNA from Drosophila melanogaster was identified, sequenced, and the model structure of the encoded polypeptide displayed a polar hydrogen-bonding network of threefold interactions, identically to the human structure. Results allow clear distinction between two subfamilies of trimeric dUTPases where altered subunit communication may account for a functional difference in the catalytic cycle.

Modeling of loops in protein structures.

Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM-22 force field, statistical preferences for the main-chain and side-chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4-, 8-, and 12-residue loop predictions, respectively, had <2 A RMSD error for the mainchain N, C(alpha), C, and O atoms; the average accuracies were 0.59 +/- 0.05, 1.16 +/- 0.10, and 2.61 +/- 0.16 A, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main-chain stem atoms is 2.5 A, the average loop prediction error increased by 180, 25, and 3% for 4-, 8-, and 12-residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.

Comparative protein structure modeling of genes and genomes.

Comparative modeling predicts the three-dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. The number of protein sequences that can be modeled and the accuracy of the predictions are increasing steadily because of the growth in the number of known protein structures and because of the improvements in the modeling software. Further advances are necessary in recognizing weak sequence-structure similarities, aligning sequences with structures, modeling of rigid body shifts, distortions, loops and side chains, as well as detecting errors in a model. Despite these problems, it is currently possible to model with useful accuracy significant parts of approximately one third of all known protein sequences. The use of individual comparative models in biology is already rewarding and increasingly widespread. A major new challenge for comparative modeling is the integration of it with the torrents of data from genome sequencing projects as well as from functional and structural genomics. In particular, there is a need to develop an automated, rapid, robust, sensitive, and accurate comparative modeling pipeline applicable to whole genomes. Such large-scale modeling is likely to encourage new kinds of applications for the many resulting models, based on their large number and completeness at the level of the family, organism, or functional network.

Core histones of the amitochondriate protist, Giardia lamblia.

Genes coding for the core histones H2a, H2b, H3, and H4 of Giardia lamblia were sequenced. A conserved organism- and gene-specific element, GRGCGCAGATTTVGG, was found upstream of the coding region in all core histone genes. The derived amino acid sequences of all four histones were similar to their homologs in other eukaryotes, although they were among the most divergent members of this protein family. Comparative protein structure modeling combined with energy evaluation of the resulting models indicated that the G. lamblia core histones individually and together can assume the same three-dimensional structures that were established by X-ray crystallography for Xenopus laevis histones and the nucleosome core particle. Since G. lamblia represents one of the earliest-diverging eukaryotes in many different molecular trees, the structure of its histones is potentially of relevance to understanding histone evolution. The G. lamblia proteins do not represent an intermediate stage between archaeal and eukaryotic histones.

Predicting the oxidation state of cysteines by multiple sequence alignment.

MOTIVATION: Protein sequences found in databanks usually do not report post translational covalent modifications such as the oxidation state of cystein (Cys) residues. Accurate prediction of whether a functionally or structurally important Cys occurs in the oxidized or thiol form would be helpful for molecular biology experiments and structure prediction. RESULTS: A new method is presented for predicting the oxidation state of Cys residues based on multiple sequence alignments and on the observation that Cys tends to occur in the same oxidation state within the same protein. The prediction of the redox state of Cys performs above 82%. The oxidation state of Cys correlates with the cellular location of the given protein within the cell, but the correlation is not perfect (up to 70%). We also perform a statistical analysis of the different redox states of Cys found in secondary structures and buried positions, and of the secondary structures linked by disulfide bonds. The results suggest that the natural borderline lies between the different oxidation states of Cys rather than between the half cystines and cysteins. AVAILABILITY: A web server implementing the prediction method is available at http://guitar.rockefeller.edu/approximately andras/cyspred.html CONTACT: fisera@rockefeller.edu

Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase.

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.

Stabilization centers in proteins: identification, characterization and predictions.

Methods are presented to locate residues, stabilization center elements, which are expected to stabilize protein structures by preventing their decay with their cooperative long range interactions. Artificial neural network-based algorithms were developed to predict these residues from the primary structure of single proteins and from the amino acid sequences of homologous proteins. The prediction accuracy using only single sequence information is 65%, but the incorporation of evolutionary information in the form of multiple alignments and conservation scores raises the efficiency by 3%. The composition, relative accessibility, number and type of interactions, conservation and the X-ray thermal factor of the identified stabilization center residues are different, not only from the whole data set but from the rest of the long range interacting residues as well. The most frequent stabilization center residues are usually found at buried positions and have a hydrophobic or aromatic side-chain, but some polar or charged residues also play an important role in the stabilization. The stabilization centers show significant difference in the composition and in the type of linked secondary structural elements compared with the rest of the residues. The performed structural and sequential conservation analysis showed the higher conservation of stabilization centers over protein families. The relation of the proposed stabilization centers to folding nuclei is also discussed.

The role of long-range interactions in defining the secondary structure of proteins is overestimated.

MOTIVATION: Secondary structure predictions based on the properties of individual residues, and sometimes on local interactions, usually fail to exceed 65% efficiency. Therefore, non-local, long-range interactions seem to be a significant cause of this limitation. RESULTS: In this paper, we apply approaches to localize highly interacting residues and clusters of residues involved in multiple non-local interactions, and test various secondary structure predictions on this separate subset to assess the effect of long-range interactions on the prediction efficiencies. It was found that only a marginal part of the failure of secondary structure predictions results from the presence of long-range interactions. Alternative possibilities are also discussed.

[Microbial contamination of small animal ambulatory veterinary clinics and waiting rooms]. / Mikrobiální kontaminace prostredí cekáren a ambulantních ordinací kliniky pro malá zvírata.

Air and contact indicator microflora was monitored for a year during 17 outpatients' examinations at indicated sites of the balance loading area, examination tables and manipulation tables in an outpatients' consulting room partitioned into two sections (I, II) and in two waiting rooms (I, II) of an internal clinic of the Faculty of Veterinary Medicine at Brno. The consulting room and both waiting rooms were after reconstruction. Ambience monitoring by a sedimentation method demonstrated the lower microbial contamination of waiting room II and outpatients' consulting room II, which were reserved for vaccination of healthy animals. The ambience in waiting room I and outpatients' consulting room I, where diseased animals were examined, showed the higher values microbial contamination. The indicators of microbial contamination under observation involved total microbial counts on meat-peptone agar (MPA), counts of hemolytic (H+) and nonhemolytic (H+) micrococci on blood agar with 10% NaCl, counts of lactose positive (L+) and lactose negative (L-) microbes on Endo agar and of molds and other microbes on Czapek-Dox agar. Frequent occurrence of representatives of the genus Aspergillus and Penicillium out of 42 genera and species of identified molds was indicated in the ambience of the waiting rooms and outpatients' consulting rooms. Examination of the indicated sites of the balance loading area in waiting room I and of examination and manipulation tables in outpatients' consulting rooms by smear and impress methods demonstrated the lowest contamination on the surface of examination tables. Microbial contamination of the balance loading area was however highest, and the identified values indicate a risk of the permanent contact of animal patients with the increased microbe concentrations during their weighing. The balance location in the waiting room is not convenient under the present schedule of cleaning and cleansing. In the Discussion section, an overall decrease in the ambience microbial contamination in waiting rooms and outpatients' consulting room under observation in building no. 9 after reconstruction is shown in comparison with the results of preceding monitoring of the waiting room and outpatients' consulting room in building no. 6 under provisional conditions.

Conservation of amino acids in multiple alignments: aspartic acid has unexpected conservation.

Analysis of the relationship between surface accessibility and amino acid conservation in multiple sequence alignments of homologous proteins confirms expected trends for hydrophobic amino acids, but reveals an unexpected difference between the conservation of Asp, Glu and Gln. Even when not in an active site, Asp is more highly conserved than Glu. There is a clear preference for conserved and buried Asp to be present in coil, but there is no tendency for Asp to conserve phi/psi in the ++ region of the Ramachandran map. Glu does not show any preference to be conserved in a particular secondary structure. Analysis of recently derived substitution matrices (e.g. BLOSUM) confirms that Glu tends to substitute more frequently with other amino acids than does Asp. Analysis of relative accessibility versus relative conservation for individual amino acid positions in alignments shows a negative correlation for all amino acid types. With the exception of Arg, Lys, Gly, Glu, Asp and Tyr, a relative conservation of > 2 suggests the amino acid will have a relative accessibility of < 50%. Observation of conserved Cys, Gly or Asp in a reliable multiple alignment suggests a position important for the structure of the protein. Furthermore, the Asp is likely to be involved in polar interactions through its side chain oxygen atoms. In contrast, Gln is the least conserved amino acid overall.

Chaos game representation of protein structures.

Chaos game representation (CGR) was proposed recently to visualize nucleotide sequences as one of the first applications of this technique in the field of biochemistry. In this paper we would like to demonstrate that representations similar to CGR can be generalized and applied for visualizing and analyzing protein databases. Examples of applications will be presented for investigating regularities, and motifs in the primary structure of proteins, and for analyzing possible structural attachments on the super-secondary structure level of proteins. A further application will be presented for testing structure prediction methods using CGR.

Different sequence environments of amino acid residues involved and not involved in long-range interactions in proteins.

No method has yet been available to decode information, hidden in the protein primary structure, on long-range interactions of amino acids. Even a limited amount of information on long-range interactions could help in conformational energy calculations of protein structures and could lead to a better understanding of how the primary structure of proteins determines their conformation. The sequence environments of amino-acid residues were compared from the viewpoint of their participation in long-range interactions. By using the simplest definition, residues were considered as partners in a long-range interaction if they were at least 20 residues apart in the sequence and their C alpha distance was less than 7 A. In spite of this rather crude definition, an analysis of 88 unrelated proteins has shown that the sequence environments (10 residues on each side) of those amino acids which are involved in long-range interactions and of those which are not are significantly different according to the criteria of mathematical statistics. Moreover, in many cases the differences are so pronounced that the involvement of a given amino acid in long-range interactions can be predicted from its sequence environment.

[Hygienic aspects of the microclimate in intensive management of rabbits]. / Hygienické aspekty mikroklimatu v intenzivním chovu králíku.

In a four-row cowshed adapted for rabbit housing, air temperatures and humidity were recorded ambulantly and instrumentally, air flow rate, cooling variable, gas content in the air, microbial contamination of air and dust deposition were determined ambulantly in the years 1991-1992 and in January to April 1993. The values of ambulant measurings show that at the outside temperature -0.5 degrees C to -5.0 degrees C the microclimate quality decreases particularly with respect to the drop of air temperature in the cowshed below 10.0 degrees C, to the increase in cooling variable up to the value 53.17 mW/cm2 and to the increase in NH3 and CO2 content to 50 ppm and/or 0.45 vol.% in absolute maximum values. In these circumstances, the average determined values of both gases are higher than the standard prescription. At outside temperatures above 27.0 degrees C the average relative air humidity in the cowshed made 69.20% for the average temperature of 25.0 degrees C. To avoid the water vapor tension exceeding the limit in the cowshed air above the value 14.1 mm Hg, when sultry atmosphere sets in, the average relative air humidity should be maximally 59.0%. Hence the cowshed was found to be insufficiently ventilated at high outside temperatures above 27.0 degrees C, and it is recommended to increase the ventilator performance and at the same time to reduce water evaporation from catchpit surfaces when urine output of rabbits is excessive as a result of the increased water intake. Temperature and air humidity readings confirmed the need of heating source installation when the outside temperatures drop below 0.0 degrees C. Evaluation of air microbial contamination showed high counts of molds and particularly of micrococci in comparison with the ambience of a cage facility for piglet raising at a repopulation station with strict hygienic regime. Microbial picture of dust deposition was positively influenced by longitudinal aerosol disinfection of the air in the cowshed.

Different sequence environments of cysteines and half cystines in proteins. Application to predict disulfide forming residues.

Protein sequences are often derived by translating genetic information, rather than by classical protein sequencing. At the DNA level cysteines and half cystines are indistinguishable. Here we show that the sequential environments of 'free' cysteine and half cystine are different. A possible origin of this difference is discussed and a simple method to predict cysteines and half cystines from the amino acid sequence is also presented.