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PubMed1999

Identification of a novel structural protein of arteriviruses.

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny virus.

Amino Acid Sequence↗

PubMed2002

Trafficking mechanism of West Nile (Sarafend) virus structural proteins.

Previous studies have shown that West Nile (Sarafend) virus matured by budding at the plasma membrane, which differs from the usual intracellular maturation of other flaviviruses. The present study investigated the trafficking mechanism of the envelope (E) and capsid (C) proteins of West Nile (Sarafend) virus during the replication cycle. The use of time-based double-immunofluorescence labelling coupled with the Triton X-100 extraction procedure revealed that both the E and C proteins were transported from the perinuclear region towards the plasma membrane along the microtubules simultaneously. The strong association of these virus proteins with the microtubules was demonstrated further with Triton X-100 extraction procedure coupled with double immunogold-labelling. Extraction of infected cells with Triton X-100 in high salt also revealed that virus E proteins were associated with the microtubules via protein-protein interaction. The disruption of microtubules with vinblastine sulphate inhibited the trafficking of both the virus E and C proteins. Both virus structural proteins were observed to co-localise and retained within vinblastine sulphate-induced microtubulin paracrystals. Extracellular virus production was also reduced drastically by vinblastine sulphate at non-cytotoxic concentration. Subsequent studies revealed that the transportation of virus E protein was associated with the microtubules-based motor protein, kinesin.

Animals↗

PubMed2005

An assessment of the accuracy of methods for predicting hydrogen positions in protein structures.

The addition of hydrogen atoms to models or experimental structures of proteins that contain only non-hydrogen atoms is a common step in crystallographic structure refinement, in theoretical studies of proteins, and in protein structure prediction. Accurate prediction of the hydrogen positions is essential, since they constitute around half of the atoms in proteins and hence contribute significantly to their energetics. Many computational tools exist for predicting hydrogen positions, although to date no quantitative comparison has been made of their accuracy or efficiency. Here we take advantage of the recent increase in ultra-high-resolution X-ray crystal structures (< 0.9 A resolution), as well as of a number of relatively high-resolution neutron diffraction structures (< 1.8 A resolution), to compare the quality of the predictions generated by a large set of commonly used methods. These include CHARMM, CNS, GROMACS, MCCE, MolProbity, WHAT IF, and X-PLOR. The hydrogen atoms that lack a rotational degree of freedom are mostly, but not always, accurately predicted. For hydrogens with a rotational degree of freedom, all the methods give much less accurate predictions. The predictions for the hydroxyl hydrogens are analyzed in detail, particularly those buried within the protein, and some explanation is provided for the errors observed. The results provide a means to make informed decisions regarding the choice and implementation of methodologies for placing hydrogens on structures of proteins. They also point to shortcomings in current force fields and suggest the need for improved descriptions of hydrogen bonding energetics.

Amines↗

PubMed1981

Biochemical, biophysical, and biological properties of densonucleosis virus (parvovirus). III. common sequences of structural proteins.

Densonucleosis virus cannot code for its four structural proteins if each of them has a unique sequence. The objective of the present investigation, therefore, was to establish whether: (i) the viral genome contains overlapping genes; (ii) the virus incorporates host proteins; or (iii) one of the structural proteins is a dimer. Two independent methods were employed for this purpose. First, the viral proteins, solubilized in sodium dodecyl sulfate, were purified after dansylation and were analyzed by peptide mapping, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, an enzyme-linked immunosorbent assay was developed for a comparative analysis of the viral proteins solubilized by sodium dodecyl sulfate. It was demonstrated with both techniques that densonucleosis virus has four unique structural proteins, all with extensive sequence homologies. Moreover, all structural proteins contained intraprotein, but no interprotein, disulfide linkages. These results indicated similarities between densonucleosis virus and representatives of the two other genera of the Parvoviridae.

Amino Acid Sequence↗

PubMed1995

The use and misuse of FTIR spectroscopy in the determination of protein structure.

Fourier transform infrared (FTIR) spectroscopy is an established tool for the structural characterization of proteins. However, many potential pitfalls exist for the unwary investigator. In this review we critically assess the application of FTIR spectroscopy to the determination of protein structure by (1) outlining the principles underlying protein secondary structure determination by FTIR spectroscopy, (2) highlighting the situations in which FTIR spectroscopy should be considered the technique of choice, (3) discussing the manner in which experiments should be conducted to derive as much physiologically relevant information as possible, and (4) outlining current methods for the determination of secondary structure from infrared spectra of proteins.

Protein Conformation↗

PubMed2004

Structure and assembly of the RNA binding domain of bluetongue virus non-structural protein 2.

Bluetongue virus non-structural protein 2 belongs to a class of highly conserved proteins found in orbiviruses of the Reoviridae family. Non-structural protein 2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. It is able to bind single-stranded RNA non-specifically, and it has been suggested that the protein is involved in the selection and condensation of the Bluetongue virus RNA segments prior to genome encapsidation. We have determined the x-ray structure of the N-terminal domain (sufficient for the RNA binding ability of non-structural protein 2) to 2.4 A resolution using anomalous scattering methods. Crystals of this apparently insoluble domain were obtained by in situ proteolysis of a soluble construct. The asymmetric unit shows two monomers related by non-crystallographic symmetry, with each monomer folded as a beta sandwich with a unique topology. The crystal structure reveals extensive monomer-monomer interactions, which explain the ability of the protein to self-assemble into large homomultimeric complexes. Of the entire surface area of the monomer, one-third is used to create the interfaces of the curved multimeric assembly observed in the x-ray structure. The structure reported here shows how the N-terminal domain would be able to bind single-stranded RNA non-specifically protecting the bound regions in a heterogeneous multimeric but not polymeric complex.

Amino Acid Sequence↗

PubMed2004

Characteristic features of amino acid residues in coiled-coil protein structures.

Detailed analyses of protein structures provide an opportunity to understand conformation and function in terms of amino acid sequence and composition. In this work, we have systematically analyzed the characteristic features of the amino acid residues found in alpha-helical coiled-coils and, in so doing, have developed indices for their properties, conformational parameters, surrounding hydrophobicity and flexibility. As expected, there is preference for hydrophobic (Ala, Leu), positive (Lys, Arg) and negatively (Glu) charged residues in coiled-coil domains. However, the surrounding hydrophobicity of residues in coiled-coil domains is significantly less than that for residues in other regions of coiled-coil proteins. The analysis of temperature factors in coiled-coil proteins shows that the residues in these domains are more stable than those in other regions. Further, we have delineated the medium- and long-range contacts in coiled-coil domains and compared the results with those obtained for other (non-coiled-coil) parts of the same proteins and non-coiled-coil helical segments of globular proteins. The residues in coiled-coil domains are largely influenced by medium-range contacts, whereas long-range interactions play a dominant role in other regions of these same proteins as well as in non-coiled-coil helices. We have also revealed the preference of amino acid residues to form cation-pi interactions and we found that Arg is more likely to form such interactions than Lys. The parameters developed in this work can be used to understand the folding and stability of coiled-coil proteins in general.

Amino Acids↗

PubMed2005

PSIbase: a database of Protein Structural Interactome map (PSIMAP).

UNLABELLED: Protein Structural Interactome map (PSIMAP) is a global interaction map that describes domain-domain and protein-protein interaction information for known Protein Data Bank structures. It calculates the Euclidean distance to determine interactions between possible pairs of structural domains in proteins. PSIbase is a database and file server for protein structural interaction information calculated by the PSIMAP algorithm. PSIbase also provides an easy-to-use protein domain assignment module, interaction navigation and visual tools. Users can retrieve possible interaction partners of their proteins of interests if a significant homology assignment is made with their query sequences. AVAILABILITY: http://psimap.org and http://psibase.kaist.ac.kr/

Binding Sites↗

PubMed2006

A multi-objective evolutionary approach to the protein structure prediction problem.

The protein structure prediction (PSP) problem is concerned with the prediction of the folded, native, tertiary structure of a protein given its sequence of amino acids. It is a challenging and computationally open problem, as proven by the numerous methodological attempts and the research effort applied to it in the last few years. The potential energy functions used in the literature to evaluate the conformation of a protein are based on the calculations of two different interaction energies: local (bond atoms) and non-local (non-bond atoms). In this paper, we show experimentally that those types of interactions are in conflict, and do so by using the potential energy function Chemistry at HARvard Macromolecular Mechanics. A multi-objective formulation of the PSP problem is introduced and its applicability studied. We use a multi-objective evolutionary algorithm as a search procedure for exploring the conformational space of the PSP problem.

Algorithms↗

PubMed1979

Amino-terminal sequence of bovine leukemia virus major internal protein: homology with mammalian type C virus p30 structural proteins.

The amino acid composition, the COOH-terminal amino acid, and the NH2-terminal amino acid sequence of the first 55 residues of the major internal structural protein, p24, of bovine leukemia virus (BLV) were determined. The compositional data and the results of end-group analysis revealed that, although BLV p24 is chemically distinct, it more closely resembles the p30 structural proteins than the other gag gene products of mammalian retroviruses. It was found that BLV p24 shares the common NH2-terminal proline and COOH-terminal leucine but lacks the common prolylleucylarginine tripeptide and the larger conserved region found near the NH2 terminus of all mammalian type C viral p30s. Alignment of the amino acid sequence of BLV p24 with the previously determined sequence of feline leukemia virus p27 revealed a statistically significant sequence homology. A more distant relationship was found between BLV p24 and other mammalian p30s. The finding of a definite sequence homology between BLV p24 and mammalian type C virus p30s clearly establishes the origin of these contemporary viral proteins from common progenitor genes.

Amino Acid Sequence↗

PubMed2006

In quest of an empirical potential for protein structure prediction.

Key to successful protein structure prediction is a potential that recognizes the native state from misfolded structures. Recent advances in empirical potentials based on known protein structures include improved reference states for assessing random interactions, sidechain-orientation-dependent pair potentials, potentials for describing secondary or supersecondary structural preferences and, most importantly, optimization protocols that sculpt the energy landscape to enhance the correlation between native-like features and the energy. Improved clustering algorithms that select native-like structures on the basis of cluster density also resulted in greater prediction accuracy. For template-based modeling, these advances allowed improvement in predicted structures relative to their initial template alignments over a wide range of target-template homology. This represents significant progress and suggests applications to proteome-scale structure prediction.

Algorithms↗

PubMed1991

Protein p22 of African swine fever virus: an early structural protein that is incorporated into the membrane of infected cells.

The open reading frame K'177, located at the left end of the African swine fever virus genome, codes for an early induced structural protein of 22,000 Da (p22), which is released from the viral particle by the nonionic detergent n-octyl-beta-D-glucopyranoside under conditions that solubilize external viral structural proteins. The predicted amino acid sequence of the protein contains a hydrophobic region at its N-terminus with characteristics of a signal peptide and, at early times after virus infection of Vero cells, the protein can be detected in the cell membrane by immunolabeling.

Amino Acid Sequence↗

PubMed1987

Cloning of a full-length complementary DNA for an Artemia salina glycine-rich protein. Structural relationship with RNA binding proteins.

Overlapping cDNAs have been isolated containing all the coding sequences for Artemia salina protein GRP33, a glycine-rich protein (16.6 mol % glycine), with a molecular weight of 32,992. GRP33 is closely related to HD40, the major protein component of Artemia heterogeneous nuclear ribonucleoprotein particles, and shares certain characteristics with other RNA binding proteins. The C-terminal region (123 amino acids) contains 39 glycine residues. This region has multiple arginine residues flanked by glycines, resembling the glycine-dimethylarginine clusters present in other RNA binding proteins. Secondary structure predictions for the protein reveal two distinct domains: a hydrophilic C-terminal domain with an extended conformation and a larger N-terminal domain with a number of alpha-helices and beta-sheets.

Amino Acid Sequence↗

PubMed1994

Three-dimensional searching for recurrent structural motifs in data bases of protein structures.

The problem of searching a data base of coordinates of proteins for substructures similar to a probe structure or motif is an important problem in computational molecular biology. It is the three-dimensional analog of the one-dimensional case of pattern matching in strings, procedures for which are widely used in molecular biology to search data bases of gene sequences. Typical applications of substructure searching are: (i) Determining whether structural features observed in one protein structure are unique or recurrent, and (ii) in predictions of protein structures, to bridge gaps in an incomplete structural model, by searching the data base for peptides that link the given starting and ending points. We describe our analysis of the problem and our experience in developing software.

Base Sequence↗

PubMed2001

Significance of aromatic-backbone amide interactions in protein structure.

Weakly polar interactions between aromatic rings of amino acids and hydrogens of backbone amides (Ar-HN) have been shown to support local structures in proteins. Their role in secondary structures, however, has not been elucidated. To investigate the relationship between Ar-HN interaction and the stability of local and secondary structures of polypeptides and to improve the prediction of this interaction based on amino acid sequence, the structures of 560 nonhomologous proteins, from the Protein Data Bank, were searched for Ar-HN interactions between the aromatic ring of each Phe, Tyr, and Trp residue at position i and the backbone amide group of any residue, except Pro, at the positions i, i - 1, i - 2, i - 3, i + 1, i + 2, and i + 3. Ar-HN interactions were identified by calculating the chemical shift of the amide hydrogen caused by the proximal aromatic ring. Ar(i)-HN(i + 1, i + 2 and i + 3) interactions were more common (7.10%, 2.08%, and 0.54%, respectively) than were Ar(i)-HN(i - 1, i - 2, and i - 3) interactions (0.66%, <0.1%, and 0.18%, respectively). The value of the chi(1) torsion angle of the aromatic residue in position i depended on the direction of the Ar-HN interaction. The position of the aromatic ring in Ar(i)-HN(i + 1, i + 2, and i + 3) interactions was mostly trans, in Ar(i)-HN(i - 1, i - 2, and i - 3) interactions mainly gauche(-), and in Ar(i)-HN(i) interactions mostly gauche(+). The analyses of the secondary structures of the protein fragments containing Ar-HN interactions showed that Ar-HN interactions were in all types of secondary structures. Search results suggest that Ar-HN interactions have a stabilizing effect on all types of secondary structures.

Amides↗

PubMed2002

A 'periodic table' for protein structures.

Current structural genomics programs aim systematically to determine the structures of all proteins coded in both human and other genomes, providing a complete picture of the number and variety of protein structures that exist. In the past, estimates have been made on the basis of the incomplete sample of structures currently known. These estimates have varied greatly (between 1,000 and 10,000; see for example refs 1 and 2), partly because of limited sample size but also owing to the difficulties of distinguishing one structure from another. This distinction is usually topological, based on the fold of the protein; however, in strict topological terms (neglecting to consider intra-chain cross-links), protein chains are open strings and hence are all identical. To avoid this trivial result, topologies are determined by considering secondary links in the form of intra-chain hydrogen bonds (secondary structure) and tertiary links formed by the packing of secondary structures. However, small additions to or loss of structure can make large changes to these perceived topologies and such subjective solutions are neither robust nor amenable to automation. Here I formalize both secondary and tertiary links to allow the rigorous and automatic definition of protein topology.

Computational Biology↗

PubMed2006

Amino acid propensities for secondary structures are influenced by the protein structural class.

Amino acid propensities for secondary structures were used since the 1970s, when Chou and Fasman evaluated them within datasets of few tens of proteins and developed a method to predict secondary structure of proteins, still in use despite prediction methods having evolved to very different approaches and higher reliability. Propensity for secondary structures represents an intrinsic property of amino acid, and it is used for generating new algorithms and prediction methods, therefore our work has been aimed to investigate what is the best protein dataset to evaluate the amino acid propensities, either larger but not homogeneous or smaller but homogeneous sets, i.e., all-alpha, all-beta, alpha-beta proteins. As a first analysis, we evaluated amino acid propensities for helix, beta-strand, and coil in more than 2000 proteins from the PDBselect dataset. With these propensities, secondary structure predictions performed with a method very similar to that of Chou and Fasman gave us results better than the original one, based on propensities derived from the few tens of X-ray protein structures available in the 1970s. In a refined analysis, we subdivided the PDBselect dataset of proteins in three secondary structural classes, i.e., all-alpha, all-beta, and alpha-beta proteins. For each class, the amino acid propensities for helix, beta-strand, and coil have been calculated and used to predict secondary structure elements for proteins belonging to the same class by using resubstitution and jackknife tests. This second round of predictions further improved the results of the first round. Therefore, amino acid propensities for secondary structures became more reliable depending on the degree of homogeneity of the protein dataset used to evaluate them. Indeed, our results indicate also that all algorithms using propensities for secondary structure can be still improved to obtain better predictive results.

Amino Acid Motifs↗

PubMed2006

Connectivity independent protein-structure alignment: a hierarchical approach.

BACKGROUND: Protein-structure alignment is a fundamental tool to study protein function, evolution and model building. In the last decade several methods for structure alignment were introduced, but most of them ignore that structurally similar proteins can share the same spatial arrangement of secondary structure elements (SSE) but differ in the underlying polypeptide chain connectivity (non-sequential SSE connectivity). RESULTS: We perform protein-structure alignment using a two-level hierarchical approach implemented in the program GANGSTA. On the first level, pair contacts and relative orientations between SSEs (i.e. alpha-helices and beta-strands) are maximized with a genetic algorithm (GA). On the second level residue pair contacts from the best SSE alignments are optimized. We have tested the method on visually optimized structure alignments of protein pairs (pairwise mode) and for database scans. For a given protein structure, our method is able to detect significant structural similarity of functionally important folds with non-sequential SSE connectivity. The performance for structure alignments with strictly sequential SSE connectivity is comparable to that of other structure alignment methods. CONCLUSION: As demonstrated for several applications, GANGSTA finds meaningful protein-structure alignments independent of the SSE connectivity. GANGSTA is able to detect structural similarity of protein folds that are assigned to different superfamilies but nevertheless possess similar structures and perform related functions, even if these proteins differ in SSE connectivity.

Algorithms↗