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PubMed1983

Identification of a poliovirus neutralization epitope through use of neutralizing antiserum raised against a purified viral structural protein.

VP4, one of the poliovirus structural proteins, was purified and used to prepare rabbit anti-VP4 serum. In addition to the anti-VP4 activity, this serum was also found to contain significant anti-VP3 and antivirion activities. The serum also effectively neutralized viral infectivity. The ease with which nonneutralizable variants were obtained indicated that neutralization was due to an antibody population which bound to a single virion epitope. Antigen saturation and immunoprecipitation experiments demonstrated that antibody to this epitope was also responsible for the serum's antivirion and anti-VP3 activities, as well as for a part of the anti-VP4 activity. The identification of a neutralization epitope most probably present on VP3, which cross-reacts with a site on denatured VP4, is the first report of such an epitope on a poliovirus structural protein other than VP1.

Antibodies, Viral↗

PubMed2001

Rosetta in CASP4: progress in ab initio protein structure prediction.

Rosetta ab initio protein structure predictions in CASP4 were considerably more consistent and more accurate than previous ab initio structure predictions. Large segments were correctly predicted (>50 residues superimposed within an RMSD of 6.5 A) for 16 of the 21 domains under 300 residues for which models were submitted. Models with the global fold largely correct were produced for several targets with new folds, and for several difficult fold recognition targets, the Rosetta models were more accurate than those produced with traditional fold recognition models. These promising results suggest that Rosetta may soon be able to contribute to the interpretation of genome sequence information.

Protein Conformation↗

PubMed2002

Evolution and physics in comparative protein structure modeling.

From a physical perspective, the native structure of a protein is a consequence of physical forces acting on the protein and solvent atoms during the folding process. From a biological perspective, the native structure of proteins is a result of evolution over millions of years. Correspondingly, there are two types of protein structure prediction methods, de novo prediction and comparative modeling. We review comparative protein structure modeling and discuss the incorporation of physical considerations into the modeling process. A good starting point for achieving this aim is provided by comparative modeling by satisfaction of spatial restraints. Incorporation of physical considerations is illustrated by an inclusion of solvation effects into the modeling of loops.

Biophysical Phenomena↗

PubMed2003

Conservation of amino acids into multiple alignments involved in pairwise interactions in three-dimensional protein structures.

We present an original strategy, that involves a bioinformatic software structure, in order to perform an exhaustive and objective statistical analysis of three-dimensional structures of proteins. We establish the relationship between multiple sequences alignments and various structural features of proteins. We show that amino acids implied in disulfide bonds, salt bridges and hydrophobic interactions have been studied. Furthermore, we point out that the more variable the sequences within a multiple alignment, the more informative the multiple alignment. The results support multiple alignments usefulness for predictions of structural features.

Amino Acid Sequence↗

PubMed2004

Structure-based functional discovery of proteins: structural proteomics.

The discovery of biochemical and cellular functions of unannotated gene products begins with a database search of proteins with structure/sequence homologues based on known genes. Very recently, a number of frontier groups in structural biology proposed a new paradigm to predict biological functions of an unknown protein on the basis of its three-dimensional structure on a genomic scale. Structural proteomics (genomics), a research area for structure-based functional discovery, aims to complete the protein-folding universe of all gene products in a cell. It would lead us to a complete understanding of a living organism from protein structure. Two major complementary experimental techniques, X-ray crystallography and NMR spectroscopy, combined with recently developed high throughput methods have played a central role in structural proteomics research; however, an integration of these methodologies together with comparative modeling and electron microscopy would speed up the goal for completing a full dictionary of protein folding space in the near future.

Animals↗

PubMed1999

Discovery of local packing motifs in protein structures.

We present a language for describing structural patterns of residues in protein structures and a method for the discovery of such patterns that recur in a set of protein structures. The patterns impose restrictions on the spatial position of each residue, their order along the amino acid chain, and which amino acids are allowed in each position. Unlike other methods for comparing sets of protein structures, our method is not based on the use of pairwise structure comparisons which is often time consuming and can produce inconsistent results. Instead, the method simultaneously takes into account information from all structures in the search for conserved structure patterns which are potential structure motifs. The method is based on describing the spatial neighborhoods of each residue in each structure as a string and applying a sequence pattern discovery method to find patterns common to subsets of these strings. Finally it is checked whether the similarities between the neighborhood strings correspond to spatially similar substructures. We apply the method to analyze sets of very disparate proteins from the four different protein families: serine proteases, cuprodoxins, cysteine proteinases, and ferredoxins. The motifs found by the method correspond well to the site and motif information given in the annotation of these proteins in PDB, Swiss-Prot, and PROSITE. Furthermore, the motifs are confirmed by using the motif data to constrain the structural alignment of the proteins obtained with the program SAP. This gave the best superposition/alignment of the proteins given the motif assignment.

Algorithms↗

PubMed2005

Nh3D: a reference dataset of non-homologous protein structures.

BACKGROUND: The statistical analysis of protein structures requires datasets in which structural features can be considered independently distributed, i.e. not related through common ancestry, and that fulfil minimal requirements regarding the experimental quality of the structures it contains. However, non-redundant datasets based on sequence similarity invariably contain distantly related homologues. Here we provide a reference dataset of non-homologous protein domains, assuming that structural dissimilarity at the topology level is incompatible with recognizable common ancestry. The dataset is based on domains at the Topology level of the CATH database which hierarchically classifies all protein structures. It contains the best refined representatives of each Topology level, validates structural dissimilarity and removes internally duplicated fragments. The compilation of Nh3D is fully scripted. RESULTS: The current Nh3D list contains 570 domains with a total of 90780 residues. It covers more than 70% of folds at the Topology level of the CATH database and represents more than 90% of the structures in the PDB that have been classified by CATH. We observe that even though all protein pairs are structurally dissimilar, some pairwise sequence identities after global alignment are greater than 30%. CONCLUSION: Nh3D is freely available as a reference dataset for the statistical analysis of sequence and structure features of proteins in the PDB. Regularly updated versions of Nh3D and the corresponding PDB-formatted coordinate sets are accessible from our Web site http://www.schematikon.org.

Algorithms↗

PubMed1998

Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles.

All eight reovirus structural proteins were resolved in a new tris, glycine, and urea (TGU) electrophoretic gel system. The specific identities of proteins were determined immunologically, biochemically, and genetically. Structural proteins of reovirus type 1 Lang had different mobilities in the TGU gel than did type 3 Dearing proteins. Intertypic reassortant viruses that contained various combinations of parental genes were used to identify each of the viral protein bands. Type 1 Lang virions were metabolically-labelled with either 3H-amino acids or 35S-methionine/cysteine and gradient purified. Aliquots of purified virions were treated to generate infectious subviral particles (ISVPs) and core particles. Radiolabelled virus, ISVP, and core proteins were resolved in the TGU gel and protein band intensities were used to determine copy numbers of each structural protein. These studies confirmed the copy numbers and locations of most reovirus proteins. However, important new findings include the discovery that virions contain approximately 120 copies of major core protein sigma 2 and 20 copies of the polymerase cofactor protein mu 2, and ISVP particles contain about 24 copies of mu 1 C that has not been processed to the delta peptide. These data are used to generate a new model of the arrangement of structural proteins with the reovirus particle.

Animals↗

PubMed2001

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.

Biophysics↗

PubMed1983

Hamster endogenous retrovirus (HaER)--distinct properties of structural proteins and DNA polymerase.

The structural proteins as well as some features of the RNA-dependent DNA polymerase of the hamster endogenous retrovirus (HaER) were examined. The polypeptide pattern of this virus is substantially different from that of other known retroviruses in containing major polypeptides with molecular weights of 68,000, 59,000, 27,000 and 24,000 daltons. Double antibody competitive radioimmunoassays showed that the HaER particles do not share any detectable antigenic relatedness with the murine viruses' p30, but manifest a considerable relatedness with the feline leukemia virus p27 and a slight cross-reactivity with the rat virus major protein. The RNA-dependent DNA polymerase of HaER virus has a molecular size of approximately 73,000 daltons and in contrast to other mammalian retroviruses shows no significant preference for Mn2+ over Mg2+. Apart from the lack of antigenic relatedness between the HaER virus proteins and the p30 protein of murine viruses, there is also no antigenic relatedness between HaER and murine viruses insofar as their DNA polymerase is concerned.

Animals↗

PubMed1988

Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

Antibodies, Monoclonal↗

PubMed1989

In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein.

Processing of dengue virus structural proteins was assessed in vitro. RNA transcripts for cell-free translation were prepared from cloned DNA (dengue virus type 4, strain 814669 genome) encoding capsid, pre-membrane (prM), and the first 23 amino acids of envelope (E). Processing of a 33-kilodalton precursor polypeptide encoded by wild-type RNA transcripts occurred only in the presence of added microsomal membranes. Under these conditions, cleavage at the capsid-prM and prM-E sites and glycosylation of prM occurred in association with translocation. Amino acid sequence analysis confirmed that translation initiated at the predicted N terminus of the capsid and that capsid-prM cleavage occurred at the predicted site for the action of signal peptidase following a candidate signal sequence (hydrophobic residues 100 to 113) in the dengue virus precursor. Mutations were introduced into the dengue virus DNA template by site-directed mutagenesis, altering nucleotide sequences encoding the capsid and the candidate signal for prM. The phenotypes of the mutants were deduced by analysis of the products of cell-free translation of the respective RNA transcripts. The resulting observations confirmed that cleavage at the capsid-prM and prM-E sites is effected entirely by signal peptidase and that the candidate signal is required for translocation.

Amino Acid Sequence↗

PubMed1999

Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon.

The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions. Both the GcvA and leucine-responsive regulatory (Lrp) proteins are required for regulation of the gcv operon. One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals. This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the I1A site from phage lambda. Integration host factor (IHF) binds the I1A site and bends the DNA about 140 degrees. Shifting the I1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent. Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered. The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase.

Amino Acid Oxidoreductases↗

PubMed1984

Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins.

We have analyzed the structure of the rubella virus genome RNA and the virus-specific RNA species synthesized in B-Vero cells infected with rubella virus. A single-stranded, capped, and polyadenylated RNA species sedimenting at 40S in a sucrose gradient was released from purified virions treated with sodium dodecyl sulfate. This RNA species migrated with an Mr of about 3.8 X 10(6) in an agarose gel after denaturation with glyoxal and dimethyl sulfoxide. Infected cells labeled with [3H]uridine in the presence of actinomycin D contained, in addition to the 40S RNA, a single-stranded polyadenylated 24S RNA species as shown by sucrose gradient analysis. In a Northern blot analysis, this RNA hybridized to a cDNA probe derived from the 3' portion of the genomic 40S RNA. In vitro translation of the 24S RNA species yielded a 110,000-dalton polypeptide, in addition to some smaller products which were immunoprecipitated with an antiserum prepared against the structural proteins E1, E2a, E2b, and C. Since the sum of the molecular weights of the nonglycosylated envelope proteins and the capsid protein has been estimated to be about 116,000 (C. Oker-Blom et al., J. Virol. 46:964-973, 1983), these results suggest that the 24S RNA species represents a subgenomic mRNA coding for a precursor (p110) to the structural proteins of rubella virus. Thus, the strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses.

Animals↗

PubMed2001

Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins.

We are studying the structural proteins and molecular interactions required for formation and release of influenza virus-like particles (VLPs) from the cell surface. To investigate these events, we generated a quadruple baculovirus recombinant that simultaneously expresses in Sf9 cells the hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 proteins of influenza virus A/Udorn/72 (H3N2). Using this quadruple recombinant, we have been able to demonstrate by double-labeling immunofluorescence that matrix protein (M1) localizes in nuclei as well as at discrete areas of the plasma membrane where HA and NA colocalize at the cell surface. Western blot analysis of cell supernatant showed that M1, HA, and NA were secreted into the culture medium. Furthermore, these proteins comigrated in similar fractions when concentrated supernatant was subjected to differential centrifugation. Electron microscopic examination (EM) of these fractions revealed influenza VLPs bearing surface projections that closely resemble those of wild-type influenza virus. Immunogold labeling and EM demonstrated that the HA and NA were present on the surface of the VLPs. We further investigated the minimal number of structural proteins necessary for VLP assembly and release using single-gene baculovirus recombinants. Expression of M1 protein alone led to the release of vesicular particles, which in gradient centrifugation analysis migrated in a similar pattern to that of the VLPs. Immunoprecipitation of M1 protein from purified M1 vesicles, VLPs, or influenza virus showed that the relative amount of M1 protein associated with M1 vesicles or VLPs was higher than that associated with virions, suggesting that particle formation and budding is a very frequent event. Finally, the HA gene within the quadruple recombinant was replaced either by a gene encoding the G protein of vesicular stomatitis virus or by a hybrid gene containing the cytoplasmic tail and transmembrane domain of the HA and the ectodomain of the G protein. Each of these constructs was able to drive the assembly and release of VLPs, although enhanced recruitment of the G glycoprotein onto the surface of the particle was observed with the recombinant carrying a G/HA chimeric gene. The described approach to assembly of wild-type and chimeric influenza VLPs may provide a valuable tool for further investigation of viral morphogenesis and genome packaging as well as for the development of novel vaccines.

Amino Acid Sequence↗

PubMed1994

Methods to study membrane protein structure in solution.

Membrane protein structure is difficult to determine by any technique. NMR spectroscopy of membrane proteins in solution can proceed using methods identical to those that have been successfully applied to numerous water-soluble proteins providing suitable solubilization conditions can be found. Organic solvents and small detergent micelles have correlation times short enough for structure determination based on 1H NOEs. Although it is difficult to generalize as each system is unique, organic solvents and micelles of strong detergents such as SDS are useful for amphiphilic peptides and small membrane proteins, whereas larger proteins need milder treatment to preserve the tertiary structure. Small unilamellar phospholipid vesicles are much too large for NOE-based structure determination, but they still fall under the domain of solution-state NMR and can be useful in certain circumstances.

Amides↗

PubMed1995

Rapid protein structure classification using one-dimensional structure profiles on the bioSCAN parallel computer.

Rapid growth of protein structures database in recent years requires an effective approach for objective comparison and classification of deposited protein structures. We describe a novel method for structure comparison and classification based on the alignment of one-dimensional structure profiles. These profiles are obtained by calculating the OCCO pseudodihedral angles (formed by O-C-C-O atoms of carbonyl groups of consecutive amino acid residues) from protein three-dimensional coordinates. These angle measurements are then converted into a 24 letter alphabet, and the protein structures are represented by sequences of letter from this alphabet. The BioSCAN parallel computer, designed for primary sequence alignment, is used to rapidly align and classify these one-dimensional structure profiles. We have developed and implemented weighted scoring matrix to identify structural classes based on commonly found structural motifs. The results of our experiments are in good agreement with the traditional protein structure classification schemes. One-dimensional structure profiles significantly improve efficiency of structure comparison and classification.

Algorithms↗

PubMed1988

In vitro assay for protein-protein interaction: carboxyl-terminal 40 residues of simian virus 40 structural protein VP3 contain a determinant for interaction with VP1.

Intermolecular interactions between polypeptide chains play essential roles in the functioning of proteins. We describe here an in vitro assay system for identifying and characterizing such interactions. Such interactions are difficult to study in vivo. We have translated synthetic, nonmethyl-capped RNAs in a cell-free protein-synthesizing system. The translation products were allowed to interact posttranslationally to form protein-protein complexes. The chemical nature of the protein interaction(s) was determined by coimmunoprecipitation of associating proteins, sedimentation through sucrose gradients, followed by NaDodSO4/polyacrylamide gel electrophoresis or by nonreducing NaDodSO4/polyacrylamide gel electrophoresis. The system has been utilized to show the self-assembly of monomeric VP1, the major structural protein of simian virus 40, into disulfide-linked pentamers and to show the noncovalent interaction of another structural protein, VP3, with VP1 at low monomer concentrations. Additionally, we show that the carboxyl-terminal 40 amino acids of VP3 are essential and sufficient for its interaction with VP1 in vitro. The in vitro assay system described here provides a method for identifying the domains involved in, and the molecular nature of, protein-protein interactions, which play an important role in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly.

Amino Acids↗