Does compactness induce secondary structure in proteins? A study of poly-alanine chains computed by distance geometry.

A few years ago, lattice model studies indicated that compactness could induce polymer chains to develop protein-like secondary structures. Subsequent off-lattice studies have found the amounts of induced structure to be relatively small. Here we use distance geometry to generate random conformations of compact poly-alanine chains of various chain lengths. The poly-alanine chains are subjected only to compactness and excluded volume constraints; no other energies or conformational propensities are included in the chain generation procedure. We find that compactness leads to considerable stabilization of secondary structure, but the absolute amount of secondary structure depends strongly on the criteria used to define helices and sheets. By loose criteria, much secondary structure arises from compactness, but by strict criteria, little does. The stabilization free energy of secondary structure provided by compactness, however, appears to be independent of criteria. Since real helices and sheets in proteins can be identified by strict criteria, we introduced small energy perturbations to compact poly-alanine chains using the AMBER force field. Small refinements produced good alpha-helices. For beta-sheets, however, larger refinements are necessary. Compactness appears to impart stability, but not much structural specificity, to secondary structures in proteins. Compactness acts more like diffusion as a force, a result of ensemble statistics, than like pair interactions such as hydrogen bonding.

Families and the structural relatedness among globular proteins.

Protein structures come in families. Are families "closely knit" or "loosely knit" entities? We describe a measure of relatedness among polymer conformations. Based on weighted distance maps, this measure differs from existing measures mainly in two respects: (1) it is computationally fast, and (2) it can compare any two proteins, regardless of their relative chain lengths or degree of similarity. It does not require finding relative alignments. The measure is used here to determine the dissimilarities between all 12,403 possible pairs of 158 diverse protein structures from the Brookhaven Protein Data Bank (PDB). Combined with minimal spanning trees and hierarchical clustering methods, this measure is used to define structural families. It is also useful for rapidly searching a dataset of protein structures for specific substructural motifs. By using an analogy to distributions of Euclidean distances, we find that protein families are not tightly knit entities.

Principles of protein folding--a perspective from simple exact models.

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.

Clinical outcomes of pyrazinamide-monoresistant Mycobacterium tuberculosis in Quebec.

BACKGROUND: In Quebec, 6.2% of all tuberculosis (TB) isolates from Canadian-born patients are resistant to pyrazinamide (PZA) alone. The clinical significance of PZA-monoresistant (PZA(MR)) TB is unknown. METHODS: Canadian-born patients with PZA(MR) TB diagnosed between 1 January 1990 and 31 December 2000 and reported in a prior study were compared to randomly selected Canadian-born patients with fully susceptible isolates diagnosed within the same time period. RESULTS: A total of 318 patients were eligible, of whom 40 (12.6%) had missing outcome information. Mean total duration of treatment was respectively 9.0 and 8.9 months for those with PZA(MR) and pan-susceptible strains. Respectively 91% and 89% of PZA(MR) and pan-susceptible patients received at least 6 months of rifampin-containing treatment. Among 67 patients with PZA(MR) TB, 51 (76%) were cured, 3 (4%) relapsed, none failed treatment, and 16 (24%) died within 6 months of diagnosis. Of 211 subjects with fully susceptible isolates, 181 (86%) were cured, 2 (1%) relapsed, 2 (1%) failed treatment, and 30 (14%) died within 6 months of diagnosis. PZA monoresistance was associated with decreased odds of successful clinical outcomes compared with pan-susceptible TB (OR 0.4, 95%CI 0.2-0.8). CONCLUSION: Patients with PZA(MR) TB had significantly worse clinical outcomes than patients with fully susceptible strains.

Automated clustering and assembly of large EST collections.

The availability of large EST (Expressed Sequence Tag) databases has led to a revolution in the way new genes are cloned. Difficulties arise, however, due to high error rates and redundancy of raw EST data. For these reasons, one of the first tasks performed by a scientist investigating any EST of interest is to gather contiguous ESTs and assemble them into a larger virtual cDNA. The REX (Recursive EST eXtender) algorithm described in this paper completely automates this process by finding ESTs that can be clustered on the basis of overlapping bases, and then assembling the contigs into a consensus sequence. By combining the clustering and assembly steps, REX can quickly generate assemblies from EST databases that are frequently updated without having to preprocess the data. A consensus assembly method is used to correct miscalled bases and remove indel errors. A unique feature of this method is that it addresses the issues of splice variants and unspliced cDNA data. Since REX is a fast greedy algorithm, it can address the problem of generating a database of assembled sequences from very large collections of EST data. A procedure is described for creating and maintaining an Assembled Consensus EST database (ACE) that is useful for characterizing the large body of data that exists in EST databases.

Mining of assembled expressed sequence tag (EST) data for protein families: application to the G protein-coupled receptor superfamily.

The availability of large expressed sequence tag (EST) databases has led to a revolution in the way new genes are identified. Mining of these databases using known protein sequences as queries is a powerful technique for discovering orthologous and paralogous genes. The scientist is often confronted, however, by an enormous amount of search output owing to the inherent redundancy of EST data. In addition, high search sensitivity often cannot be achieved using only a single member of a protein superfamily as a query. In this paper a technique for addressing both of these issues is described. Assembled EST databases are queried with every member of a protein superfamily, the results are integrated and false positives are pruned from the set. The result is a set of assemblies enriched in members of the protein superfamily under consideration. The technique is applied to the G protein-coupled receptor (GPCR) superfamily in the construction of a GPCR Resource. A novel full-length human GPCR identified from the GPCR Resource is presented, illustrating the utility of the method.

Cloning and characterization of human protease-activated receptor 4.

Protease-activated receptors 1-3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47/Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By fluorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.