How Effective Is The Data On Co-Occurrence Of Domains In Multi-Domain Proteins In Prediction Of Protein-Protein Interactions?

We explore the fuse of information on co-occurrence of domains in multi-domain proteins in predicting protein-protein interactions. The basic premise of our work is the assumption that domains co-occurring in a polypeptide chain undergo either structural or functional interactions among themselves. In this study we use a template dataset of domains in multidomain proteins and predict protein-protein interactions in a target organism. We note that maximum number of correct predictions of interacting protein domain families (158) is made in S. cerevisiae when the dataset of closely related organisms is used as the template followed by the more diverse dataset of bacterial proteins (48) and a dataset of randomly chosen proteins (23). We conclude that use of multi-domain information from organisms closely-related to the target can aid prediction of interacting protein families.

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.

Dynamic nature of proteins - interpretation of residual dipolar couplings

Protein conformations and dynamics can be studied by nuclear magnetic resonance spectroscopy using dilute liquid crystalline samples. This work clarifies the interpretation of residual dipolar coupling data yielded by the experiments. It was discovered that unfolded proteins without any additional structure beyond that of a mere polypeptide chain exhibit residual dipolar couplings. Also, it was found that molecular dynamics induce fluctuations in the molecular alignment and doing so affect residual dipolar couplings. The finding clarified the origins of low order parameter values observed earlier. The work required the development of new analytical and computational methods for the prediction of intrinsic residual dipolar coupling profiles for unfolded proteins. The presented characteristic chain model is able to reproduce the general trend of experimental residual dipolar couplings for denatured proteins. The details of experimental residual dipolar coupling profiles are beyond the analytical model, but improvements are proposed to achieve greater accuracy. A computational method for rapid prediction of unfolded protein residual dipolar couplings was also developed. Protein dynamics were shown to modulate the effective molecular alignment in a dilute liquid crystalline medium. The effects were investigated from experimental and molecular dynamics generated conformational ensembles of folded proteins. It was noted that dynamics induced alignment is significant especially for the interpretation of molecular dynamics in small, globular proteins. A method of correction was presented. Residual dipolar couplings offer an attractive possibility for the direct observation of protein conformational preferences and dynamics. The presented models and methods of analysis provide significant advances in the interpretation of residual dipolar coupling data from proteins.

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

The phylogeny and evolutionary timescale of muscoide (Diptera: Brachycera: Calyptratae) inferred from mitochondrial genomes

Muscoidea is a significant dipteran clade that includes house flies (Family Muscidae), latrine flies (F. Fannidae), dung flies (F. Scathophagidae) and root maggot flies (F. Anthomyiidae). It is comprised of approximately 7000 described species. The monophyly of the Muscoidea and the precise relationships of muscoids to the closest superfamily the Oestroidea (blow flies, flesh flies etc) are both unresolved. Until now mitochondrial (mt) genomes were available for only two of the four muscoid families precluding a thorough test of phylogenetic relationships using this data source. Here we present the first two mt genomes for the families Fanniidae (Euryomma sp.) (family Fanniidae) and Anthomyiidae (Delia platura (Meigen, 1826)). We also conducted phylogenetic analyses containing of these newly sequenced mt genomes plus 15 other species representative of dipteran diversity to address the internal relationship of Muscoidea and its systematic position. Both maximum-likelihood and Bayesian analyses suggested that Muscoidea was not a monophyletic group with the relationship: (Fanniidae + Muscidae) + ((Anthomyiidae + Scathophagidae) + (Calliphoridae + Sarcophagidae)), supported by the majority of analysed datasets. This also infers that Oestroidea was paraphyletic in the majority of analyses. Divergence time estimation suggested that the earliest split within the Calyptratae, separating (Tachinidae + Oestridae) from the remaining families, occurred in the Early Eocene. The main divergence within the paraphyletic muscoidea grade was between Fanniidae + Muscidae and the lineage ((Anthomyiidae + Scathophagidae) + (Calliphoridae + Sarcophagidae)) which occurred in the Late Eocene

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin.

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.

Conformations of disulfide bridges in proteins

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of 2.0 Å, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (φ, Ψ) values at Cys residues, bridge dihedral angles χss, χ1i, χ1j, χ2i and χ2j the distances Cαi-Cαj and Cβi-Cβj between the Cα and Cβ atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson ((1981) Adv. Protein Chem. 34, 167–330). Disulfide bridging across antiparallel extended strands is observed in α-lytic protease, crambin, and β-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.

Peptide mimics for structural features in proteins. Crystal structures of three heptapeptide helices with a C-terminal 6-->1 hydrogen bond.

The crystal structure determination of three heptapeptides containing alpha-aminoisobutyryl (Aib) residues as a means of helix stabilization provides a high-resolution characterization of 6-->1 hydrogen-bonded conformations, reminiscent of helix-terminating structural features in proteins. The crystal parameters for the three peptides, Boc-Val-Aib-X-Aib-Ala-Aib-Y-OMe, where X and Y are Phe, Leu (I), Leu, Phe (II) and Leu, Leu (III) are: (I) space group P1, Z = 1, a = 9.903 A, b = 10.709 A, c = 11.969 A, alpha = 102.94 degrees, beta = 103.41 degrees, gamma = 92.72 degrees, R = 4.55%; (II) space group P21, Z = 2, a = 10.052 A, b = 17.653 A, c = 13.510 A, beta = 108.45 degrees, R = 4.49%; (III) space group P1, Z = 2 (two independent molecules IIIa and IIIb in the asymmetric unit), a = 10.833 A, b = 13.850 A, c = 16.928 A, alpha = 99.77 degrees, beta = 105.90 degrees, gamma = 90.64 degrees, R = 8.54%. In all cases the helices form 3(10)/alpha-helical (or 3(10)helical) structures, with helical columns formed by head-to-tail hydrogen bonding. The helices assemble in an all-parallel motif in crystals I and III and in an antiparallel motif in II. In the four crystallographically characterized molecules, I, II, IIIa and IIIb, Aib(6) adopts a left-handed helical (hL) conformation with positive phi, psi values, resulting in 6-->1 hydrogen-bond formation between Aib(2) CO and Leu(7)/Phe(7) NH groups. In addition a 4-->1 hydrogen bond is seen between Aib(3) CO and Aib(6) NH groups. This pattern of hydrogen bonding is often observed at the C-terminus of helices proteins, with the terminal pi-type turn being formed by four residues adopting the hRhRhRhL conformation.

Conformational characteristics of asparaginyl residues in proteins

Backbone conformations at 1064 asparaginyl residues in 123 non-homologous, high-resolution X-ray structures of proteins were analysed. Asn adopts conformations in left-handed x-helical region and other partially allowed regions in the Ramachandran map more readily than any other non-glycyl residue. Asn conformational clusters in the (phi,psi) regions of left-handed alpha-helix, right-handed alpha-helix and extended (beta) strands were investigated in detail for their occurrence in various secondary structures, especially in beta-turn regions. Preferences were observed for Asn conformations in different positions in various beta-turn types, including the first and fourth positions of the turn. Asparaginyl residues with extended conformations are found to occur frequently in irregular regions, although they are expected to occur predominantly in extended strands or in the third position of type II beta-turns. Asn conformations at the N-cap positions of helices strongly prefer extended conformation than alpha(L), which seems to be characteristic of non-glycyl residues at that position. In the linkers connecting two extended strands and those connecting an alpha-helix and an extended strand, Asn with alpha(L) or alpha(R) conformation is more favoured than Asn with the beta-conformation. Analysis of Asn-Asn doublets and Asn-X-Asn triplets permitted identification of conformational families in such sequences. Results of this investigation provide useful hints in modelling Asn-rich regions in proteins such as malaria parasite coat protein. (C) Munksgaard 1994.

Disc electrophoresis of sorghum seed proteins in polyacrylamide gels

Electrophoretic analyses of sorghum flour protein by disc electrophoresis in polyacrylamide gels containing urea have been described. The albumin, globulin, and prolamin fractions of sorghum endosperm meal have been investigated, using pH 9.5 and 4.3 gel systems with four different buffers. Highly complex patterns were observed for all three protein fractions. It has been suggested that this method can provide a convenient tool for the analyses of seed proteins which are relatively insoluble in aqueous buffers.