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Sample records for functional protein hinge

  1. Dynamic requirements for a functional protein hinge.

    PubMed

    Kempf, James G; Jung, Ju-Yeon; Ragain, Christina; Sampson, Nicole S; Loria, J Patrick

    2007-04-20

    The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11 residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a tenfold higher Km value and 10(3)-fold reduced k(cat) relative to WT with either d-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates <750 s(-1) that are an order of magnitude slower than the natural WT loop 6 motion. At the same time, nanosecond timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by 1H, 15N and 13CO chemical-shifts, the glycine substitutions diminished the enzyme's response to ligand, and induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of WT. These data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.

  2. Approximation by hinge functions

    SciTech Connect

    Faber, V.

    1997-05-01

    Breiman has defined {open_quotes}hinge functions{close_quotes} for use as basis functions in least squares approximations to data. A hinge function is the max (or min) function of two linear functions. In this paper, the author assumes the existence of smooth function f(x) and a set of samples of the form (x, f(x)) drawn from a probability distribution {rho}(x). The author hopes to find the best fitting hinge function h(x) in the least squares sense. There are two problems with this plan. First, Breiman has suggested an algorithm to perform this fit. The author shows that this algorithm is not robust and also shows how to create examples on which the algorithm diverges. Second, if the author tries to use the data to minimize the fit in the usual discrete least squares sense, the functional that must be minimized is continuous in the variables, but has a derivative which jumps at the data. This paper takes a different approach. This approach is an example of a method that the author has developed called {open_quotes}Monte Carlo Regression{close_quotes}. (A paper on the general theory is in preparation.) The author shall show that since the function f is continuous, the analytic form of the least squares equation is continuously differentiable. A local minimum is solved for by using Newton`s method, where the entries of the Hessian are estimated directly from the data by Monte Carlo. The algorithm has the desirable properties that it is quadratically convergent from any starting guess sufficiently close to a solution and that each iteration requires only a linear system solve.

  3. Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR.

    PubMed

    Swint-Kruse, Liskin; Larson, Christopher; Pettitt, B Montgomery; Matthews, Kathleen Shive

    2002-04-01

    LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.

  4. Engineering upper hinge improves stability and effector function of a human IgG1.

    PubMed

    Yan, Boxu; Boyd, Daniel; Kaschak, Timothy; Tsukuda, Joni; Shen, Amy; Lin, Yuwen; Chung, Shan; Gupta, Priyanka; Kamath, Amrita; Wong, Anne; Vernes, Jean-Michel; Meng, Gloria Y; Totpal, Klara; Schaefer, Gabriele; Jiang, Guoying; Nogal, Bartek; Emery, Craig; Vanderlaan, Martin; Carter, Paul; Harris, Reed; Amanullah, Ashraf

    2012-02-17

    Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

  5. Effect of the hinge protein on the heme iron site of cytochrome c sub 1

    SciTech Connect

    Kim, C.H.; Yencha, A.J.; Bunker, G.; Zhang, G.; Chance, B.; King, T.E. )

    1989-02-21

    X-ray absorption spectroscopic (XAS) studies on cytochrome c{sub 1} from beef heart mitochondria were conducted to identify the effect of the hinge protein on the structure of the heme site in cytochrome c{sub 1}. A comparison of XAS data of highly purified one-band and two-band cytochrome c{sub 1} demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c{sub 1}: a conformational change occurs within a radius of approximately 5 {angstrom} from the heme iron in cytochrome c{sub 1} when the hinge protein is bound to cytochrome c{sub 1}. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c{sub 1} are affected by the hinge protein.

  6. Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

    PubMed Central

    Sun, J.; Sampson, N. S.

    1998-01-01

    We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families. PMID:9684881

  7. Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

    PubMed

    Sun, J; Sampson, N S

    1998-07-01

    We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.

  8. Mechanism of Epac activation: structural and functional analyses of Epac2 hinge mutants with constitutive and reduced activities.

    PubMed

    Tsalkova, Tamara; Blumenthal, Donald K; Mei, Fang C; White, Mark A; Cheng, Xiaodong

    2009-08-28

    Epac2 is a member of the family of exchange proteins directly activated by cAMP (Epac). Our previous studies suggest a model of Epac activation in which cAMP binding to the enzyme induces a localized "hinge" motion that reorients the regulatory lobe relative to the catalytic lobe without inducing large conformational changes within individual lobes. In this study, we identified the location of the major hinge in Epac2 by normal mode motion correlation and structural alignment analyses. Targeted mutagenesis was then performed to test the functional importance of hinge bending for Epac activation. We show that substitution of the conserved residue phenylalanine 435 with glycine (F435G) facilitates the hinge bending and leads to a constitutively active Epac2 capable of stimulating nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan (F435W), impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters determined by small angle x-ray scattering further reveal that whereas the F435G mutant assumes a more extended conformation in the absence of cAMP, the F435W mutant is incapable of adopting the fully extended and active conformation in the presence of cAMP. These findings demonstrate the importance of hinge motion in Epac activation. Our study also suggests that phenylalanine at position 435 is the optimal size side chain to keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac extension and activation in the presence of cAMP.

  9. Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Sun, J; Sampson, N S

    1999-08-31

    In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

  10. The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis.

    PubMed

    Dey, D; Oleinikov, A V; Traut, R R

    1995-01-01

    A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12: delta 42-52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917-922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12:(Pro)7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:delta 42-52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:delta 42-52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bound in the A site with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro7 sequence retains sufficient flexibility for full activity.

  11. Mutations on the hinge region of leukocyte elastase inhibitor determine the loss of inhibitory function.

    PubMed

    Perani, P; Zeggai, S; Torriglia, A; Courtois, Y

    2000-08-11

    Leukocyte elastase inhibitor (LEI) is a cytosolic component of lung macrophages and blood leukocytes that inhibits neutrophil elastase. LEI is a member of the serpin superfamily, these proteins, mostly protease inhibitors, are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the hinge region of the reactive centre loop into a beta-sheet of the inhibitor. In this work three mutations were produced in the hinge region of elastase inhibitor that abolish the inhibition activity of LEI and transform the protein in a substrate of the elastase. This result demonstrates that the inhibitory mechanism of serpin is common to LEI.

  12. Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration.

    PubMed

    Sim, Jaehyun; Sim, Jun; Park, Eunsung; Lee, Julian

    2015-06-01

    Many proteins undergo large-scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non-overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter-domain bending motion. The performance of the algorithm is demonstrated on several proteins.

  13. Design of Bioelectronic Interfaces by Exploiting Hinge-Bending Motions in Proteins

    NASA Astrophysics Data System (ADS)

    Benson, David E.; Conrad, David W.; de Lorimier, Robert M.; Trammell, Scott A.; Hellinga, Homme W.

    2001-08-01

    We report a flexible strategy for transducing ligand-binding events into electrochemical responses for a wide variety of proteins. The method exploits ligand-mediated hinge-bending motions, intrinsic to the bacterial periplasmic binding protein superfamily, to establish allosterically controlled interactions between electrode surfaces and redox-active, Ru(II)-labeled proteins. This approach allows the development of protein-based bioelectronic interfaces that respond to a diverse set of analytes. Families of these interfaces can be generated either by exploiting natural binding diversity within the superfamily or by reengineering the specificity of individual proteins. These proteins may have numerous medical, environmental, and defense applications.

  14. Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

    PubMed

    Bade, Veronika N; Nickels, Jochen; Keusekotten, Kirstin; Praefcke, Gerrit J K

    2012-01-01

    The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent

  15. Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

    SciTech Connect

    Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.; Erb, Steven M.; Luy, Betty E.; Calvert, Amanda E.; Blair, Carol D.; Roehrig, John T.; Huang, Claire Y.-H.

    2011-04-25

    Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.

  16. Encephalomyocarditis virus Leader protein hinge domain is responsible for interactions with Ran GTPase

    SciTech Connect

    Bacot-Davis, Valjean R.; Palmenberg, Ann C.

    2013-08-15

    Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35–40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition. - Highlights: • The hinge domain provides critical residues in Cardiovirus L:Ran complex formation. • Leader prefers to bind Ran in a nucleotide free, GDP-conformation. • L-induced Nup62 phosphorylation is reduced with Ran-deficient binding mutations.

  17. De novo design of protein kinase inhibitors by in silico identification of hinge region-binding fragments.

    PubMed

    Urich, Robert; Wishart, Grant; Kiczun, Michael; Richters, André; Tidten-Luksch, Naomi; Rauh, Daniel; Sherborne, Brad; Wyatt, Paul G; Brenk, Ruth

    2013-05-17

    Protein kinases constitute an attractive family of enzyme targets with high relevance to cell and disease biology. Small molecule inhibitors are powerful tools to dissect and elucidate the function of kinases in chemical biology research and to serve as potential starting points for drug discovery. However, the discovery and development of novel inhibitors remains challenging. Here, we describe a structure-based de novo design approach that generates novel, hinge-binding fragments that are synthetically feasible and can be elaborated to small molecule libraries. Starting from commercially available compounds, core fragments were extracted, filtered for pharmacophoric properties compatible with hinge-region binding, and docked into a panel of protein kinases. Fragments with a high consensus score were subsequently short-listed for synthesis. Application of this strategy led to a number of core fragments with no previously reported activity against kinases. Small libraries around the core fragments were synthesized, and representative compounds were tested against a large panel of protein kinases and subjected to co-crystallization experiments. Each of the tested compounds was active against at least one kinase, but not all kinases in the panel were inhibited. A number of compounds showed high ligand efficiencies for therapeutically relevant kinases; among them were MAPKAP-K3, SRPK1, SGK1, TAK1, and GCK for which only few inhibitors are reported in the literature.

  18. Encephalomyocarditis virus Leader protein hinge domain is responsible for interactions with Ran GTPase.

    PubMed

    Bacot-Davis, Valjean R; Palmenberg, Ann C

    2013-08-15

    Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35-40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition.

  19. Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor.

    PubMed

    Shaffer, Paul L; McDonnell, Donald P; Gewirth, Daniel T

    2005-02-22

    The vitamin D receptor (VDR) is a ligand-responsive transcription factor that forms active, heterodimeric complexes with the 9-cis retinoic acid receptor (RXR) on vitamin D response elements (VDREs). Both proteins consist of an N-terminal DNA-binding domain, a C-terminal ligand-binding domain, and an intervening hinge region. The length requirements of the hinge for both transcriptional regulation and DNA binding have not been studied to date for any member of the steroid hormone superfamily. We have generated a series of internal deletion mutants of the VDR hinge and found that deletion of as few as five amino acids from the C-terminus of the hinge significantly reduces transcriptional activation in vivo. Replacing deleted residues in the C-terminus of the hinge with alanines restored activity, indicating that this section of the hinge acts as a sequence-independent spacer. The hinge region of VDR forms a long helix, and the geometric consequences of this structure may explain the requirement of the hinge region for transcriptional activity. Interestingly, all of the deletion mutants, even those that do not activate transcription, bind VDREs with equal and high affinity, indicating that the defect in these mutants is not their ability to bind VDREs. In contrast to VDR, constructs of RXR containing deletions of up to 14 amino acids in the hinge region exhibit near wild-type transcriptional activity. The ability to delete more of the RXR hinge may be related to the additional plasticity required by its role as the common heterodimer partner for nuclear receptors on differing DNA elements.

  20. The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase.

    PubMed

    Xiang, J; Sun, J; Sampson, N S

    2001-04-01

    We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase. The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations. The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502. Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity. They all contain Pro at the first position. Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate. However, the reduced catalytic activities observed are not due to increased rates of loop opening. To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 "double-hinge" mutants. The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another. It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme. The independence of intermediate release on amino acid sequence is consistent with the absence of a "universal" hinge sequence in structurally related enzymes. PMID:11286559

  1. The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase.

    PubMed

    Xiang, J; Sun, J; Sampson, N S

    2001-04-01

    We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase. The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations. The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502. Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity. They all contain Pro at the first position. Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate. However, the reduced catalytic activities observed are not due to increased rates of loop opening. To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 "double-hinge" mutants. The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another. It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme. The independence of intermediate release on amino acid sequence is consistent with the absence of a "universal" hinge sequence in structurally related enzymes.

  2. The impact of joint line restoration on functional results after hinged knee prosthesis

    PubMed Central

    Yilmaz, Serdar; Cankaya, Deniz; Deveci, Alper; Firat, Ahmet; Ozkurt, Bulent; Bozkurt, Murat

    2016-01-01

    Background: Hinged knee prosthesis is an effective treatment method as a salvage procedure in marked ligamentous insufficiency and severe bone defects. Joint line determination and restoration are difficult due to large bone defects and distorted anatomy. We evaluated the impact of joint line alteration on the outcome in rotating hinge knee arthroplasty (RHKA). Materials and Methods: 35 patients who had rotating hinged knee prosthesis applied between 2008 and 2013 were evaluated in this retrospective study. The patients were studied radiologically and clinically. Five patients were lost to followup and two patients died, leaving a total of 28 (7 male, 21 female) patients for final evaluation. The average age of the patients was 66.19 ± 8.35 years (range 52–83 years). The patients were evaluated clinically with Knee Society knee and functional score and patellar score. The joint line positions were evaluated radiographically with femoral epicondylar ratio method. The outcomes were also evaluated according to age, body weight and gender. Student's t-test, independent t-test, and the Wilcoxon signed rank test were used in the statistical analysis. Results: The mean Knee Society knee and functional score significantly improved from preoperative 19.52 ± 11.77 and 12.5 ± 15.66 respectively to 72.46 ± 14.01 and 70.36 ± 9.22 respectively postoperatively (P < 0.001). The mean range of motion of the knee improved from 55.95° ± 25.08° preoperatively to 92.14° ± 13.47° postoperatively (P < 0.001). Joint line position was restored in 20 patients (71.4%). Joint line alteration did not affect Knee Society Scores (KSSs) in contrast to patellar scores. Additionally, KSS was better in the patients with body mass index ≤30 at followup (P = 0.022 and P = 0.045). Conclusion: RHKA is an effective salvage procedure for serious instability and large bone defects. Restoration of the joint line improves the patellar score although it had no effect on the clinical outcome

  3. A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins

    PubMed Central

    Kim, Hanseong; Zou, Taisong; Modi, Chintan; Dörner, Katerina; Grunkemeyer, Timothy J.; Chen, Liqing; Fromme, Raimund; Matz, Mikhail V.; Ozkan, S. Banu; Wachter, Rebekka M.

    2015-01-01

    Summary In proteins, functional divergence involves mutations that modify structure and dynamics. Here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-tored photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities. PMID:25565105

  4. A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins.

    PubMed

    Kim, Hanseong; Zou, Taisong; Modi, Chintan; Dörner, Katerina; Grunkemeyer, Timothy J; Chen, Liqing; Fromme, Raimund; Matz, Mikhail V; Ozkan, S Banu; Wachter, Rebekka M

    2015-01-01

    In proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.

  5. Hinge assembly

    DOEpatents

    Vandergriff, D.H.

    1999-08-31

    A hinge assembly is disclosed having a first leaf, a second leaf and linking member. The first leaf has a contact surface. The second leaf has a first contact surface and a second contact surface. The linking member pivotally connects to the first leaf and to the second leaf. The hinge assembly is capable of moving from a closed position to an open position. In the closed position, the contact surface of the first leaf merges with the first contact surface of the second leaf. In the open position, the contact surface of the first leaf merges with the second contact surface of the second leaf. The hinge assembly can include a seal on the contact surface of the first leaf. 8 figs.

  6. Hinge assembly

    DOEpatents

    Vandergriff, David Houston

    1999-01-01

    A hinge assembly having a first leaf, a second leaf and linking member. The first leaf has a contact surface. The second leaf has a first contact surface and a second contact surface. The linking member pivotally connects to the first leaf and to the second leaf. The hinge assembly is capable of moving from a closed position to an open position. In the closed position, the contact surface of the first leaf merges with the first contact surface of the second leaf. In the open position, the contact surface of the first leaf merges with the second contact surface of the second leaf. The hinge assembly can include a seal on the contact surface of the first leaf.

  7. Role of the interdomain hinge of flavocytochrome b2 in intra- and inter-protein electron transfer.

    PubMed

    Sharp, R E; White, P; Chapman, S K; Reid, G A

    1994-05-01

    The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a hinge peptide. Kinetics experiments [White, P., Manson, F. D. C., Brunt, C. E., Chapman, S. K., & Reid, GA. (1993) Biochem. J. 291, 89-94] have illustrated the importance for efficient interdomain electron transfer of maintaining the structural integrity of the hinge. To probe the role of the hinge in a more subtle manner, we have constructed a mutant enzyme, H delta 3, which has a three amino acid deletion in the hinge region. Intra- and inter-protein electron transfer within H delta 3 flavocytochrome b2 and the H delta 3:cytochrome c redox complex was investigated by steady-state and stopped-flow kinetics analysis. The H delta 3 mutant enzyme remains a good L-lactate dehydrogenase, as is evident from steady-state experiments with ferricyanide as electron acceptor (40% less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (15% less active than wild-type enzyme). The global effect of the deletion is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of the H delta 3 enzyme is manifested at two electron-transfer steps on the catalytic cycle of flavocytochrome b2. First, the rate of heme reduction has fallen 5-fold in H delta 3 compared with the wild-type enzyme (from 445 to 91 s-1), due to poor interdomain electron transfer from flavin to heme.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Kursula, I; Salin, M; Sun, J; Norledge, B V; Haapalainen, A M; Sampson, N S; Wierenga, R K

    2004-04-01

    The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed. PMID:15166315

  9. Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Kursula, I; Salin, M; Sun, J; Norledge, B V; Haapalainen, A M; Sampson, N S; Wierenga, R K

    2004-04-01

    The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.

  10. Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function.

    PubMed

    Nascimento, Alessandro S; Dias, Sandra Martha Gomes; Nunes, Fábio M; Aparício, Ricardo; Ambrosio, Andre L B; Bleicher, Lucas; Figueira, Ana Carolina M; Santos, Maria Auxiliadora M; de Oliveira Neto, Mário; Fischer, Hannes; Togashi, Marie; Craievich, Aldo F; Garratt, Richard C; Baxter, John D; Webb, Paul; Polikarpov, Igor

    2006-07-14

    The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.

  11. A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies.

    PubMed

    Vermersch, P S; Tesmer, J J; Lemon, D D; Quiocho, F A

    1990-09-25

    The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.

  12. A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation.

    PubMed

    Kanno, Tatsuo; Bucher, Etienne; Daxinger, Lucia; Huettel, Bruno; Böhmdorfer, Gudrun; Gregor, Wolfgang; Kreil, David P; Matzke, Marjori; Matzke, Antonius J M

    2008-05-01

    RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.

  13. Multi-functional hinge equipped with a magneto-rheological rotary damper for solar array deployment system

    NASA Astrophysics Data System (ADS)

    Wen, Mingfu; Yu, Miao; Fu, Jie; Wu, Zhengzhong

    2015-02-01

    This article describes the design and simulation of a novel multi-functional hinge equipped with a rotary magnetorheological damper for solar array deployment system, which is comprised of a hinge, an angular sensor, a positioning and locking mechanism and a rotary damper. In order to achieve the compact design in structure, some components were reused in different function modules. It's the first to use magnet-rheological fluid (MRF) to dissipate the energy in solar array deployment system. The main advantage in using MR rotary damper instead of a viscous fluid rotary damper is that the damping force of MR damper can be adjusted according to the external magnetic field environment excited. A mechanic model was built and the structure design was focused on the MR rotary damper, a damping force model of this damper is deduced based on hydromechanics with Bingham plastic constitutive model. A simulation of deployment motion was taken to validate the motion sequence of various components during the unfolding and locking process. It can be obtained that a constant damping coefficient can hardly balance the different performance of solar deployment system, then a simulation of the proposed deployment system equipped with rotary MR damper was carried out. According to the simulation, it can be obtained that the terminal velocity decreased by 75.81% and the deployment time decreased by 72.37% compared with a given constant damping coefficients. Therefore, the proposed new type of rotary damper can reach a compromise with different performance utilizing an on-off control strategy.

  14. Immobilization of the distal hinge in the labile serpin plasminogen activator inhibitor 1: identification of a transition state with distinct conformational and functional properties.

    PubMed

    De Taeye, Bart; Compernolle, Griet; Dewilde, Maarten; Biesemans, Wouter; Declerck, Paul J

    2003-06-27

    The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsin-susceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of alphahF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of alphahF, subsequently resulting in substrate behavior.

  15. Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions.

    PubMed

    Kurihara, Makoto; Ohmuro-Matsuyama, Yuki; Ayabe, Keiichi; Yamashita, Takahiro; Yamaji, Hideki; Ueda, Hiroshi

    2016-01-01

    Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.

  16. Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

    PubMed

    Messer, William B; de Alwis, Ruklanthi; Yount, Boyd L; Royal, Scott R; Huynh, Jeremy P; Smith, Scott A; Crowe, James E; Doranz, Benjamin J; Kahle, Kristen M; Pfaff, Jennifer M; White, Laura J; Sariol, Carlos A; de Silva, Aravinda M; Baric, Ralph S

    2014-02-01

    The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens. PMID:24385585

  17. Gene Duplication, Lineage-Specific Expansion, and Subfunctionalization in the MADF-BESS Family Patterns the Drosophila Wing Hinge

    PubMed Central

    Shukla, Vallari; Habib, Farhat; Kulkarni, Apurv; Ratnaparkhi, Girish S.

    2014-01-01

    Gene duplication, expansion, and subsequent diversification are features of the evolutionary process. Duplicated genes can be lost, modified, or altered to generate novel functions over evolutionary timescales. These features make gene duplication a powerful engine of evolutionary change. In this study, we explore these features in the MADF-BESS family of transcriptional regulators. In Drosophila melanogaster, the family contains 16 similar members, each containing an N-terminal, DNA-binding MADF domain and a C-terminal, protein-interacting, BESS domain. Phylogenetic analysis shows that members of the MADF-BESS family are expanded in the Drosophila lineage. Three members, which we name hinge1, hinge2, and hinge3 are required for wing development, with a critical role in the wing hinge. hinge1 is a negative regulator of Winglesss expression and interacts with core wing-hinge patterning genes such as teashirt, homothorax, and jing. Double knockdowns along with heterologous rescue experiments are used to demonstrate that members of the MADF-BESS family retain function in the wing hinge, in spite of expansion and diversification for over 40 million years. The wing hinge connects the blade to the thorax and has critical roles in fluttering during flight. MADF-BESS family genes appear to retain redundant functions to shape and form elements of the wing hinge in a robust and fail-safe manner. PMID:24336749

  18. Valgus osteotomy for hinge abduction.

    PubMed

    de Gheldere, Antoine; Eastwood, Deborah M

    2011-07-01

    Failure of the enlarged and deformed anterolateral portion of the femoral head to roll into the acetabulum during abduction alters hip joint mechanics. The resultant hinge abduction is associated with pain, and the patient often has restricted movement. A valgus osteotomy removes the deformed portion of the femoral head away from the weight-bearing area and ensures there is pain-free congruent range of movement around the weight-bearing position. The concomitant improvement in lever arm function and leg length results in a better gait pattern. In immature patients, abolition of hinge abduction allows the lateral acetabular ossification center to grow more normally. PMID:21742146

  19. Deletion of the regulatory domain of protein kinase C alpha exposes regions in the hinge and catalytic domains that mediate nuclear targeting.

    PubMed

    James, G; Olson, E

    1992-02-01

    Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha-transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the

  20. Designing Great Hinge Questions

    ERIC Educational Resources Information Center

    Wiliam, Dylan

    2015-01-01

    According to author Dylan Wiliam, because lessons never go exactly as planned, teachers should build plan B into plan A. This involves designing a lesson with a "hinge" somewhere in the middle and using specific kinds of questions--what he calls hinge questions--to quickly assess students' understanding of a concept before moving on.…

  1. Damped leaf flexure hinge

    NASA Astrophysics Data System (ADS)

    Chen, Zhong; Chen, Guisheng; Zhang, Xianmin

    2015-05-01

    Flexure-based mechanism like compliant actuation system embeds complex dynamics that will reduce the control bandwidth and limits their dynamic positioning precision. This paper presents a theoretical model of a leaf flexure hinge with damping layers using strain energy method and Kelvin damping model. The modified loss factor of the damped leaf flexure hinge is derived, and the equivalent viscous damping coefficient of the damped leaf hinge is obtained, which could be used to improve the pseudo-rigid-model. The free vibration signals of the hinge in three different damping configurations are measured. The experimental modal analysis also is performed on the three kinds of damped leaf flexure hinges in order to evaluate their 1st order bending natural frequency and vibration-suppressing effects. The evaluation of modified loss factor model also is performed. The experimental results indicate that the constrained layer damping can enhance the structure damping of the hinge even if only single damping layer each side, the modified loss factor model can get good predicts of a damped leaf flexure hinge in the frequency range below 1st order natural frequency, and it is necessary that the dimensional parameters of the damping layers and basic layer of the hinge should be optimized for simplification at the mechanism's design stage.

  2. Damped leaf flexure hinge.

    PubMed

    Chen, Zhong; Chen, Guisheng; Zhang, Xianmin

    2015-05-01

    Flexure-based mechanism like compliant actuation system embeds complex dynamics that will reduce the control bandwidth and limits their dynamic positioning precision. This paper presents a theoretical model of a leaf flexure hinge with damping layers using strain energy method and Kelvin damping model. The modified loss factor of the damped leaf flexure hinge is derived, and the equivalent viscous damping coefficient of the damped leaf hinge is obtained, which could be used to improve the pseudo-rigid-model. The free vibration signals of the hinge in three different damping configurations are measured. The experimental modal analysis also is performed on the three kinds of damped leaf flexure hinges in order to evaluate their 1st order bending natural frequency and vibration-suppressing effects. The evaluation of modified loss factor model also is performed. The experimental results indicate that the constrained layer damping can enhance the structure damping of the hinge even if only single damping layer each side, the modified loss factor model can get good predicts of a damped leaf flexure hinge in the frequency range below 1st order natural frequency, and it is necessary that the dimensional parameters of the damping layers and basic layer of the hinge should be optimized for simplification at the mechanism's design stage. PMID:26026549

  3. Ligand- and coactivator-mediated transactivation function (AF2) of the androgen receptor ligand-binding domain is inhibited by the cognate hinge region.

    PubMed

    Wang, Q; Lu, J; Yong, E L

    2001-03-01

    Transactivation functions (AF2) in the ligand-binding domains (LBD) of many steroid receptors are well characterized, but there is little evidence to support such a function for the LBD of the androgen receptor (AR). We report a mutant AR, with residues 628-646 in the hinge region deleted, which exhibited transactivation activity that was more than double that of the wild type (WT) AR. Although no androgen-dependent AF2 activity could be observed for the WT ARLBD fused to a heterologous DNA-binding domain, the mutant ARLBD(Delta628-646) was 30-40 times more active than the WT ARLBD. In the presence of the p160 coactivator TIF2, AR(Delta628-646) was significantly more active than similarly treated WT AR. Deletion of residues 628-646 also enhanced TIF2-ARLBD activity 8-fold, an effect not present when the LBD-interacting LXXLL motifs of TIF2 were mutated, suggesting that the negative modulatory activity of residues 628-646 were exerted via coactivator pathways. Although the AP-1 (c-Jun/c-Fos) system and NcoR have been reported to interact with and repress the activity of some steroid receptors, c-Jun, c-Fos, c-Jun/c-Fos, nor NcoR function was consistently affected by the absence or presence of residues 628-646, implying that the AR hinge region exerts its silencing effects in a manner independent of these corepressors. Our data provide evidence for the novel finding that strong androgen-dependent AF2 exists in the ARLBD and is the first report of a negative regulatory domain in the AR. Because mutations in this region are commonly associated with prostate cancer, it is important to characterize the mechanisms by which the hinge region exerts its repressor effect on ligand-activated and coactivator-mediated AF2 activity of the ARLBD.

  4. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  5. The hinge as a spacer contributes to covalent assembly and is required for function of IgG.

    PubMed

    Coloma, M J; Trinh, K R; Wims, L A; Morrison, S L

    1997-01-15

    Chimeric IgG3 lacking a genetic hinge but with Cys residues in CH2 between position 231 and 232 (IgG3 deltaH+Cys) and position 279 (IgG3 deltaH+2Cys) produced by site-directed mutagenesis have been found to be deficient in their intermolecular assembly. Although some H2L2 molecules are formed, significant quantities of assembly intermediates, in particular HL, are found in the secretions. Both IgG3 deltaH+Cys and IgG3 deltaH+2Cys were greatly reduced in their ability to bind Fc gammaRI. They also failed to bind C1q and activate the complement cascade. Addition of the tailpiece from IgM (microtp) to IgG3 deltaH+Cys (yielding IgG3 deltaH+Cys microtp) failed to lead to efficient oligomerization. IgG3 deltaH+Cys microtp failed to bind Fc gammaRII. However, at high concentrations it was able to bind C1q and effect complement-dependent cytolysis. It consumed complement in the absence of added Ag, indicating that removing the flexible hinge was not sufficient to yield polymeric IgG that resembled IgM in its dependence on added Ag for complement activation.

  6. Elliptical flexure hinges

    NASA Astrophysics Data System (ADS)

    Smith, Stuart T.; Badami, Vivek G.; Dale, Jami S.; Xu, Ying

    1997-03-01

    This paper presents closed form equations based on a modification of those originally derived by Paros and Weisbord in 1965, for the mechanical compliance of a simple monolithic flexure hinge of elliptic cross section, the geometry of which is determined by the ratio ɛ of the major and minor axes. It is shown that these equations converge at ɛ=1 to the Paros and Weisbord equations for a hinge of circular section and at ɛ ⇒∞ to the equations predicted from simple beam bending theory for the compliance of a cantilever beam. These equations are then assessed by comparison with results from finite element analysis over a range of geometries typical of many hinge designs. Based on the finite element analysis, stress concentration factors for the elliptical hinge are also presented. As a further verification of these equations, a number of elliptical hinges were manufactured on a CNC milling machine. Experimental data were produced by applying a bending moment using dead weight loading and measuring subsequent angular deflections with a laser interferometer. In general, it was found that predictions for the compliance of elliptical hinges are likely to be within 12% for a range of geometries with the ratio βx(=t/2ax) between 0.06 and 0.2 and for values of ɛ between 1 and 10.

  7. Notch flexure hinges: An effective theory

    NASA Astrophysics Data System (ADS)

    Tseytlin, Yakov M.

    2002-09-01

    This article presents effective tractable equations for rotational compliance (stiffness) of a simple monolithic flexure hinge with circular (radius R and crosspiece thickness t), elliptical [at semiaxis ax, ay, elliptical ratio epsilon (=ax/ay)] and other cross sections. These equations and the method by inverse conformal mapping of circular approximating contour used to derive them are different from the known and widely used theoretical equations originally derived in 1965 by Paros and Weisbord for circular notch hinges. Later it was found that the circular hinge represents the worst case error between known theoretical and finite element models. The conformal mapping equations data presented in this article are likely to be much closer (within less than 10%) to the finite element analysis and experimental data than other theoretical equations. In particular this is the case for circular notch hinges at relative thickness beta(=t/2R) in the range 0.01 to 0.3 and for elliptical hinges at the elliptical ratio epsilon=1 to 10. The derived general equation is common for all types of notch hinges whose profiles can be approximated by two shifted contiguous circles and includes material parameters with Young's modulus and Poisson's ratio. The latter is totally omitted in known theoretical solutions by other authors. New tractable equations are derived from the general equation on the basis of trigonometric functions' simplified series expansion in certain ranges of hinge crosspiece relative thickness. The corresponding graphs are presented. Experimental data were received by holographic interference and autocollimator measurement.

  8. Backlash-Free Locking Hinge

    NASA Technical Reports Server (NTRS)

    Wessekski, Clarence J.

    1987-01-01

    Tight joints achieved without precisely machined parts. Hinge for foldable structures locked with minimum force by human operator. Once locked, hinge makes strong, tight joint. Loose fit, or joint slop, common to commercial locking hinges eliminated. Despite tight fit, new hinge concept does not impose close tolerances on manufacture of its parts. Developed for erecting unfoldable structures in space, hinge used on collapsible scaffolds and similar terrestrial structures.

  9. Functional Protein Microarray Technology

    PubMed Central

    Hu, Shaohui; Xie, Zhi; Qian, Jiang; Blackshaw, Seth; Zhu, Heng

    2010-01-01

    Functional protein microarrays are emerging as a promising new tool for large-scale and high-throughput studies. In this article, we will review their applications in basic proteomics research, where various types of assays have been developed to probe binding activities to other biomolecules, such as proteins, DNA, RNA, small molecules, and glycans. We will also report recent progress of using functional protein microarrays in profiling protein posttranslational modifications, including phosphorylation, ubiquitylation, acetylation, and nitrosylation. Finally, we will discuss potential of functional protein microarrays in biomarker identification and clinical diagnostics. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology. PMID:20872749

  10. Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.

    PubMed

    Sharff, A J; Rodseth, L E; Spurlino, J C; Quiocho, F A

    1992-11-10

    The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.

  11. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  12. Protective hinge in insulin opens to enable its receptor engagement.

    PubMed

    Menting, John G; Yang, Yanwu; Chan, Shu Jin; Phillips, Nelson B; Smith, Brian J; Whittaker, Jonathan; Wickramasinghe, Nalinda P; Whittaker, Linda J; Pandyarajan, Vijay; Wan, Zhu-li; Yadav, Satya P; Carroll, Julie M; Strokes, Natalie; Roberts, Charles T; Ismail-Beigi, Faramarz; Milewski, Wieslawa; Steiner, Donald F; Chauhan, Virander S; Ward, Colin W; Weiss, Michael A; Lawrence, Michael C

    2014-08-19

    Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function. PMID:25092300

  13. A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo.

    PubMed

    Brezski, Randall J; Kinder, Michelle; Grugan, Katharine D; Soring, Keri L; Carton, Jill; Greenplate, Allison R; Petley, Theodore; Capaldi, Dorie; Brosnan, Kerry; Emmell, Eva; Watson, Sharon; Jordan, Robert E

    2014-01-01

    We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')₂ fragments, but not for full-length IgG or for closely-related F(ab')₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')₂ fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')₂ fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is

  14. Shape Memory Composite Hybrid Hinge

    NASA Technical Reports Server (NTRS)

    Fang, Houfei; Im, Eastwood; Lin, John; Scarborough, Stephen

    2012-01-01

    There are two conventional types of hinges for in-space deployment applications. The first type is mechanically deploying hinges. A typical mechanically deploying hinge is usually composed of several tens of components. It is complicated, heavy, and bulky. More components imply higher deployment failure probability. Due to the existence of relatively moving components among a mechanically deploying hinge, it unavoidably has microdynamic problems. The second type of conventional hinge relies on strain energy for deployment. A tape-spring hinge is a typical strain energy hinge. A fundamental problem of a strain energy hinge is that its deployment dynamic is uncontrollable. Usually, its deployment is associated with a large impact, which is unacceptable for many space applications. Some damping technologies have been experimented with to reduce the impact, but they increased the risks of an unsuccessful deployment. Coalescing strain energy components with shape memory composite (SMC) components to form a hybrid hinge is the solution. SMCs are well suited for deployable structures. A SMC is created from a high-performance fiber and a shape memory polymer resin. When the resin is heated to above its glass transition temperature, the composite becomes flexible and can be folded or packed. Once cooled to below the glass transition temperature, the composite remains in the packed state. When the structure is ready to be deployed, the SMC component is reheated to above the glass transition temperature, and it returns to its as-fabricated shape. A hybrid hinge is composed of two strain energy flanges (also called tape-springs) and one SMC tube. Two folding lines are placed on the SMC tube to avoid excessive strain on the SMC during folding. Two adapters are used to connect the hybrid hinge to its adjacent structural components. While the SMC tube is heated to above its glass transition temperature, a hybrid hinge can be folded and stays at folded status after the temperature

  15. 14 CFR 23.657 - Hinges.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Hinges. 23.657 Section 23.657 Aeronautics... Surfaces § 23.657 Hinges. (a) Control surface hinges, except ball and roller bearing hinges, must have a... material used as a bearing. (b) For ball or roller bearing hinges, the approved rating of the bearing...

  16. 14 CFR 23.657 - Hinges.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Hinges. 23.657 Section 23.657 Aeronautics... Surfaces § 23.657 Hinges. (a) Control surface hinges, except ball and roller bearing hinges, must have a... material used as a bearing. (b) For ball or roller bearing hinges, the approved rating of the bearing...

  17. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    PubMed

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  18. 14 CFR 25.657 - Hinges.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Hinges. 25.657 Section 25.657 Aeronautics... STANDARDS: TRANSPORT CATEGORY AIRPLANES Design and Construction Control Surfaces § 25.657 Hinges. (a) For control surface hinges, including ball, roller, and self-lubricated bearing hinges, the approved rating...

  19. 14 CFR 25.657 - Hinges.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Hinges. 25.657 Section 25.657 Aeronautics... STANDARDS: TRANSPORT CATEGORY AIRPLANES Design and Construction Control Surfaces § 25.657 Hinges. (a) For control surface hinges, including ball, roller, and self-lubricated bearing hinges, the approved rating...

  20. Plastic hinge modeling of structures

    NASA Astrophysics Data System (ADS)

    Maruthayappan, Ramakrishnan

    1994-07-01

    The rapid changes taking place in vehicle design has resulted in a great variety of vehicles having different structural configurations. The behavior of such structures under impact or crash conditions demand an efficient modeling of the system. Since a vehicle crash is a dynamic phenomenon exhibiting complex interaction between structural and inertial forces, the model should be able to predict the transient deformations which range from small strain elastic deformations to large strain plastic deformations. Among various formulations and modeling techniques available, the plastic hinge theory offers a simple, realistic and a computationally efficient model. This thesis explores and applies the plastic hinge modeling technique to some simple structures ranging from an elementary cantilever beam to a torque box representing a passenger compartment. The nonlinear static and dynamic behavior of a general aviation seat model for different load cases have been predicted using this theory. A detailed study and application of nonlinear finite element technique has also been performed. A comparative study of the responses yielded by plastic hinge model and finite element model demonstrates the effectiveness and accuracy of the plastic hinge model in predicting the behavior of the structures reliably. This thesis also studies and predicts the dynamics of mechanical systems using plastic hinges modeled with flexible and rigid bodies with contact-impact and plastic deformation.

  1. High accuracy flexural hinge development

    NASA Astrophysics Data System (ADS)

    Santos, I.; Ortiz de Zárate, I.; Migliorero, G.

    2005-07-01

    This document provides a synthesis of the technical results obtained in the frame of the HAFHA (High Accuracy Flexural Hinge Assembly) development performed by SENER (in charge of design, development, manufacturing and testing at component and mechanism levels) with EADS Astrium as subcontractor (in charge of doing an inventory of candidate applications among existing and emerging projects, establishing the requirements and perform system level testing) under ESA contract. The purpose of this project has been to develop a competitive technology for a flexural pivot, usuable in highly accurate and dynamic pointing/scanning mechanisms. Compared with other solutions (e.g. magnetic or ball bearing technologies) flexural hinges are the appropriate technology for guiding with accuracy a mobile payload over a limited angular ranges around one rotation axes.

  2. Functions of S100 Proteins

    PubMed Central

    Donato, R.; Cannon, B.R.; Sorci, G.; Riuzzi, F.; Hsu, K.; Weber, D.J.; Geczy, C.L.

    2013-01-01

    The S100 protein family consists of 24 members functionally distributed into three main subgroups: those that only exert intracellular regulatory effects, those with intracellular and extracellular functions and those which mainly exert extracellular regulatory effects. S100 proteins are only expressed in vertebrates and show cell-specific expression patterns. In some instances, a particular S100 protein can be induced in pathological circumstances in a cell type that does not express it in normal physiological conditions. Within cells, S100 proteins are involved in aspects of regulation of proliferation, differentiation, apoptosis, Ca2+ homeostasis, energy metabolism, inflammation and migration/invasion through interactions with a variety of target proteins including enzymes, cytoskeletal subunits, receptors, transcription factors and nucleic acids. Some S100 proteins are secreted or released and regulate cell functions in an autocrine and paracrine manner via activation of surface receptors (e.g. the receptor for advanced glycation end-products and toll-like receptor 4), G-protein-coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and N-glycans. Extracellular S100A4 and S100B also interact with epidermal growth factor and basic fibroblast growth factor, respectively, thereby enhancing the activity of the corresponding receptors. Thus, extracellular S100 proteins exert regulatory activities on monocytes/macrophages/microglia, neutrophils, lymphocytes, mast cells, articular chondrocytes, endothelial and vascular smooth muscle cells, neurons, astrocytes, Schwann cells, epithelial cells, myoblasts and cardiomyocytes, thereby participating in innate and adaptive immune responses, cell migration and chemotaxis, tissue development and repair, and leukocyte and tumor cell invasion. PMID:22834835

  3. Alternative S2 Hinge Regions of the Myosin Rod Affect Myofibrillar Structure and Myosin Kinetics

    SciTech Connect

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.

    2009-07-01

    The subfragment 2/light meromyosin 'hinge' region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.

  4. Learning Protein Folding Energy Functions

    PubMed Central

    Guan, Wei; Ozakin, Arkadas; Gray, Alexander; Borreguero, Jose; Pandit, Shashi; Jagielska, Anna; Wroblewska, Liliana; Skolnick, Jeffrey

    2014-01-01

    A critical open problem in ab initio protein folding is protein energy function design, which pertains to defining the energy of protein conformations in a way that makes folding most efficient and reliable. In this paper, we address this issue as a weight optimization problem and utilize a machine learning approach, learning-to-rank, to solve this problem. We investigate the ranking-via-classification approach, especially the RankingSVM method and compare it with the state-of-the-art approach to the problem using the MINUIT optimization package. To maintain the physicality of the results, we impose non-negativity constraints on the weights. For this we develop two efficient non-negative support vector machine (NNSVM) methods, derived from L2-norm SVM and L1-norm SVMs, respectively. We demonstrate an energy function which maintains the correct ordering with respect to structure dissimilarity to the native state more often, is more efficient and reliable for learning on large protein sets, and is qualitatively superior to the current state-of-the-art energy function. PMID:25311546

  5. Predicting protein functions from PPI networks using functional aggregation.

    PubMed

    Hou, Jingyu; Chi, Xiaoxiao

    2012-11-01

    Predicting protein functions computationally from massive protein-protein interaction (PPI) data generated by high-throughput technology is one of the challenges and fundamental problems in the post-genomic era. Although there have been many approaches developed for computationally predicting protein functions, the mutual correlations among proteins in terms of protein functions have not been thoroughly investigated and incorporated into existing prediction methods, especially in voting based prediction methods. In this paper, we propose an innovative method to predict protein functions from PPI data by aggregating the functional correlations among relevant proteins using the Choquet-Integral in fuzzy theory. This functional aggregation measures the real impact of each relevant protein function on the final prediction results, and reduces the impact of repeated functional information on the prediction. Accordingly, a new protein similarity and a new iterative prediction algorithm are proposed in this paper. The experimental evaluations on real PPI datasets demonstrate the effectiveness of our method.

  6. Phospholipid liposomes functionalized by protein

    NASA Astrophysics Data System (ADS)

    Glukhova, O. E.; Savostyanov, G. V.; Grishina, O. A.

    2015-03-01

    Finding new ways to deliver neurotrophic drugs to the brain in newborns is one of the contemporary problems of medicine and pharmaceutical industry. Modern researches in this field indicate the promising prospects of supramolecular transport systems for targeted drug delivery to the brain which can overcome the blood-brain barrier (BBB). Thus, the solution of this problem is actual not only for medicine, but also for society as a whole because it determines the health of future generations. Phospholipid liposomes due to combination of lipo- and hydrophilic properties are considered as the main future objects in medicine for drug delivery through the BBB as well as increasing their bioavailability and toxicity. Liposomes functionalized by various proteins were used as transport systems for ease of liposomes use. Designing of modification oligosaccharide of liposomes surface is promising in the last decade because it enables the delivery of liposomes to specific receptor of human cells by selecting ligand and it is widely used in pharmacology for the treatment of several diseases. The purpose of this work is creation of a coarse-grained model of bilayer of phospholipid liposomes, functionalized by specific to the structural elements of the BBB proteins, as well as prediction of the most favorable orientation and position of the molecules in the generated complex by methods of molecular docking for the formation of the structure. Investigation of activity of the ligand molecule to protein receptor of human cells by the methods of molecular dynamics was carried out.

  7. Hinged-Blade, Vertical-Shaft Windmill

    NASA Technical Reports Server (NTRS)

    Shultz, B., Jr.

    1985-01-01

    Vertical-shaft windmill concept calls for hinged, flapping blades to increase energy-conversion efficiency by reducing wind-energy loss. Hinged Blade Halves unfold to catch wind when moving with it, then fold away from wind when moving against it.

  8. Multifunctional Deployment Hinges Rigidified by Ultraviolet

    NASA Technical Reports Server (NTRS)

    Kerslake, Thomas W.; Simburger, Edward J.; Matusmoto, James; Giants, Thomas W.; Garcia, Alexander; Perry, Alan; Rawal, Suraj; Marshall, Craig; Lin, John Kun Hung; Day, Jonathan Robert; Scarborough, Stephen Emerson

    2005-01-01

    Multifunctional hinges have been developed for deploying and electrically connecting panels comprising planar arrays of thin-film solar photovoltaic cells. In the original intended application of these hinges, the panels would be facets of a 32-sided (and approximately spherical) polyhedral microsatellite (see figure), denoted a PowerSphere, that would be delivered to orbit in a compact folded configuration, then deployed by expansion of gas in inflation bladders. Once deployment was complete, the hinges would be rigidified to provide structural connections that would hold the panels in their assigned relative positions without backlash. Such hinges could also be used on Earth for electrically connecting and structurally supporting solar panels that are similarly shipped in compact form and deployed at their destinations. As shown in section A-A in the figure, a hinge of this type is partly integrated with an inflation bladder and partly integrated with the frame of a solar panel. During assembly of the hinge, strip extensions from a flexible circuit harness on the bladder are connected to corresponding thin-film conductors on the solar panel by use of laser welding and wrap-around contacts. The main structural component of the hinge is a layer of glass fiber impregnated with an ultraviolet-curable resin. After deployment, exposure to ultraviolet light from the Sun cures the resin, thereby rigidifying the hinge.

  9. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    PubMed

    Sours, Kevin M; Xiao, Yao; Ahn, Natalie G

    2014-05-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.

  10. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  11. A new protein structure representation for efficient protein function prediction.

    PubMed

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  12. New protein functions in yeast chromosome VIII.

    PubMed Central

    Ouzounis, C.; Bork, P.; Casari, G.; Sander, C.

    1995-01-01

    The analysis of the 269 open reading frames of yeast chromosome VIII by computational methods has yielded 24 new significant sequence similarities to proteins of known function. The resulting predicted functions include three particularly interesting cases of translation-associated proteins: peptidyl-tRNA hydrolase, a ribosome recycling factor homologue, and a protein similar to cytochrome b translational activator CBS2. The methodological limits of the meaningful transfer of functional information between distant homologues are discussed. PMID:8563640

  13. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  14. 14 CFR 23.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Loads parallel to hinge line. 23.393... Control Surface and System Loads § 23.393 Loads parallel to hinge line. (a) Control surfaces and supporting hinge brackets must be designed to withstand inertial loads acting parallel to the hinge line....

  15. 14 CFR 23.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Loads parallel to hinge line. 23.393... Control Surface and System Loads § 23.393 Loads parallel to hinge line. (a) Control surfaces and supporting hinge brackets must be designed to withstand inertial loads acting parallel to the hinge line....

  16. 14 CFR 25.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Loads parallel to hinge line. 25.393... § 25.393 Loads parallel to hinge line. (a) Control surfaces and supporting hinge brackets must be designed for inertia loads acting parallel to the hinge line. (b) In the absence of more rational data,...

  17. 14 CFR 25.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Loads parallel to hinge line. 25.393... § 25.393 Loads parallel to hinge line. (a) Control surfaces and supporting hinge brackets must be designed for inertia loads acting parallel to the hinge line. (b) In the absence of more rational data,...

  18. Protein Function Prediction: Problems and Pitfalls.

    PubMed

    Pearson, William R

    2015-01-01

    The characterization of new genomes based on their protein sets has been revolutionized by new sequencing technologies, but biologists seeking to exploit new sequence information are often frustrated by the challenges associated with accurately assigning biological functions to newly identified proteins. Here, we highlight some of the challenges in functional inference from sequence similarity. Investigators can improve the accuracy of function prediction by (1) being conservative about the evolutionary distance to a protein of known function; (2) considering the ambiguous meaning of "functional similarity," and (3) being aware of the limitations of annotations in functional databases. Protein function prediction does not offer "one-size-fits-all" solutions. Prediction strategies work better when the idiosyncrasies of function and functional annotation are better understood. PMID:26334923

  19. Protein Function Prediction: Problems and Pitfalls.

    PubMed

    Pearson, William R

    2015-01-01

    The characterization of new genomes based on their protein sets has been revolutionized by new sequencing technologies, but biologists seeking to exploit new sequence information are often frustrated by the challenges associated with accurately assigning biological functions to newly identified proteins. Here, we highlight some of the challenges in functional inference from sequence similarity. Investigators can improve the accuracy of function prediction by (1) being conservative about the evolutionary distance to a protein of known function; (2) considering the ambiguous meaning of "functional similarity," and (3) being aware of the limitations of annotations in functional databases. Protein function prediction does not offer "one-size-fits-all" solutions. Prediction strategies work better when the idiosyncrasies of function and functional annotation are better understood.

  20. Origins of Protein Functions in Cells

    NASA Technical Reports Server (NTRS)

    Seelig, Burchard; Pohorille, Andrzej

    2011-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known

  1. Year 2 Report: Protein Function Prediction Platform

    SciTech Connect

    Zhou, C E

    2012-04-27

    Upon completion of our second year of development in a 3-year development cycle, we have completed a prototype protein structure-function annotation and function prediction system: Protein Function Prediction (PFP) platform (v.0.5). We have met our milestones for Years 1 and 2 and are positioned to continue development in completion of our original statement of work, or a reasonable modification thereof, in service to DTRA Programs involved in diagnostics and medical countermeasures research and development. The PFP platform is a multi-scale computational modeling system for protein structure-function annotation and function prediction. As of this writing, PFP is the only existing fully automated, high-throughput, multi-scale modeling, whole-proteome annotation platform, and represents a significant advance in the field of genome annotation (Fig. 1). PFP modules perform protein functional annotations at the sequence, systems biology, protein structure, and atomistic levels of biological complexity (Fig. 2). Because these approaches provide orthogonal means of characterizing proteins and suggesting protein function, PFP processing maximizes the protein functional information that can currently be gained by computational means. Comprehensive annotation of pathogen genomes is essential for bio-defense applications in pathogen characterization, threat assessment, and medical countermeasure design and development in that it can short-cut the time and effort required to select and characterize protein biomarkers.

  2. Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)*

    PubMed Central

    Plomp, Rosina; Dekkers, Gillian; Rombouts, Yoann; Visser, Remco; Koeleman, Carolien A.M.; Kammeijer, Guinevere S.M.; Jansen, Bas C.; Rispens, Theo; Hensbergen, Paul J.; Vidarsson, Gestur; Wuhrer, Manfred

    2015-01-01

    Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes. PMID:25759508

  3. Protein function prediction based on data fusion and functional interrelationship.

    PubMed

    Meng, Jun; Wekesa, Jael-Sanyanda; Shi, Guan-Li; Luan, Yu-Shi

    2016-04-01

    One of the challenging tasks of bioinformatics is to predict more accurate and confident protein functions from genomics and proteomics datasets. Computational approaches use a variety of high throughput experimental data, such as protein-protein interaction (PPI), protein sequences and phylogenetic profiles, to predict protein functions. This paper presents a method that uses transductive multi-label learning algorithm by integrating multiple data sources for classification. Multiple proteomics datasets are integrated to make inferences about functions of unknown proteins and use a directed bi-relational graph to assign labels to unannotated proteins. Our method, bi-relational graph based transductive multi-label function annotation (Bi-TMF) uses functional correlation and topological PPI network properties on both the training and testing datasets to predict protein functions through data fusion of the individual kernel result. The main purpose of our proposed method is to enhance the performance of classifier integration for protein function prediction algorithms. Experimental results demonstrate the effectiveness and efficiency of Bi-TMF on multi-sources datasets in yeast, human and mouse benchmarks. Bi-TMF outperforms other recently proposed methods. PMID:26869536

  4. Phosphoinositide Control of Membrane Protein Function

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Zhang, Miao; Mahajan, Rahul; Meng, Xuan-Yu; Adney, Scott K.; Cui, Meng; Baki, Lia

    2015-01-01

    Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca2+]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states. PMID:25293526

  5. Graphite Composite Booms with Integral Hinges

    NASA Technical Reports Server (NTRS)

    Alexander, Wes; Carlos, Rene; Rossoni, Peter; Sturm, James

    2006-01-01

    A document discusses lightweight instrument booms under development for use aboard spacecraft. A boom of this type comprises a thin-walled graphite fiber/ matrix composite tube with an integral hinge that can be bent for stowage and later allowed to spring back to straighten the boom for deployment in outer space. The boom design takes advantage of both the stiffness of the composite in tubular geometry and the flexibility of thin sections of the composite. The hinge is formed by machining windows in the tube at diametrically opposite locations so that there remain two opposing cylindrical strips resembling measuring tapes. Essential to the design is a proprietary composite layup that renders the hinge tough yet flexible enough to be bendable as much as 90 in either of two opposite directions. When the boom is released for deployment, the torque exerted by the bent hinge suffices to overcome parasitic resistance from harnesses and other equipment, so that the two sections of the hinge snap to a straight, rigid condition in the same manner as that of measuring tapes. Issues addressed in development thus far include selection of materials, out-of-plane bending, edge cracking, and separation of plies.

  6. J domain independent functions of J proteins.

    PubMed

    Ajit Tamadaddi, Chetana; Sahi, Chandan

    2016-07-01

    Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

  7. Food Protein Functionality--A New Model.

    PubMed

    Foegeding, E Allen

    2015-12-01

    Proteins in foods serve dual roles as nutrients and structural building blocks. The concept of protein functionality has historically been restricted to nonnutritive functions--such as creating emulsions, foams, and gels--but this places sole emphasis on food quality considerations and potentially overlooks modifications that may also alter nutritional quality or allergenicity. A new model is proposed that addresses the function of proteins in foods based on the length scale(s) responsible for the function. Properties such as flavor binding, color, allergenicity, and digestibility are explained based on the structure of individual molecules; placing this functionality at the nano/molecular scale. At the next higher scale, applications in foods involving gelation, emulsification, and foam formation are based on how proteins form secondary structures that are seen at the nano and microlength scales, collectively called the mesoscale. The macroscale structure represents the arrangements of molecules and mesoscale structures in a food. Macroscale properties determine overall product appearance, stability, and texture. The historical approach of comparing among proteins based on forming and stabilizing specific mesoscale structures remains valid but emphasis should be on a common means for structure formation to allow for comparisons across investigations. For applications in food products, protein functionality should start with identification of functional needs across scales. Those needs are then evaluated relative to how processing and other ingredients could alter desired molecular scale properties, or proper formation of mesoscale structures. This allows for a comprehensive approach to achieving the desired function of proteins in foods.

  8. Magnetic Actuation of Self-Assembled DNA Hinges

    NASA Astrophysics Data System (ADS)

    Lauback, S.; Mattioli, K.; Armstrong, M.; Miller, C.; Pease, C.; Castro, C.; Sooryakumar, R.

    DNA nanotechnology offers a broad range of applications spanning from the creation of nanoscale devices, motors and nanoparticle templates to the development of precise drug delivery systems. Central to advancing this technology is the ability to actuate or reconfigure structures in real time, which is currently achieved primarily by DNA strand displacement yielding slow actuation times (about 1-10min). Here we exploit superparamagnetic beads to magnetically actuate DNA structures which also provides a system to measure forces associated with molecular interactions. DNA nanodevices are folded using DNA origami, whereby a long single-stranded DNA is folded into a precise compact geometry using hundreds of short oligonucleotides. Our DNA nanodevice is a nanohinge from which rod shaped DNA nanostructures are polymerized into micron-scale filaments forming handles for actuation. By functionalizing one arm of the hinge and the filament ends, the hinge can be attached to a surface while still allowing an arm to rotate and the filaments can be labeled with magnetic beads enabling the hinge to be actuated almost instantaneously by external magnetic fields. These results lay the groundwork to establish real-time manipulation and direct force application of DNA constructs.

  9. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    SciTech Connect

    Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2009-12-18

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

  10. Atomic-level functional model of dengue virus Envelope protein infectivity.

    PubMed

    Christian, Elizabeth A; Kahle, Kristen M; Mattia, Kimberly; Puffer, Bridget A; Pfaff, Jennifer M; Miller, Adam; Paes, Cheryl; Davidson, Edgar; Doranz, Benjamin J

    2013-11-12

    A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity.

  11. Control-surface hinge-moment calculations for a high-aspect-ratio supercritical wing

    NASA Technical Reports Server (NTRS)

    Perry, B., III

    1978-01-01

    The hinge moments, at selected flight conditions, resulting from deflecting two trailing edge control surfaces (one inboard and one midspan) on a high aspect ratio, swept, fuel conservative wing with a supercritical airfoil are estimated. Hinge moment results obtained from procedures which employ a recently developed transonic analysis are given. In this procedure a three dimensional inviscid transonic aerodynamics computer program is combined with a two dimensional turbulent boundary layer program in order to obtain an interacted solution. These results indicate that trends of the estimated hinge moment as a function of deflection angle are similar to those from experimental hinge moment measurements made on wind tunnel models with swept supercritical wings tested at similar values of free stream Mach number and angle of attack.

  12. Comparison of hinged and contoured rods for occipitocervical arthrodesis in adults: A clinical study

    PubMed Central

    Abode-Iyamah, Kingsley O; Dlouhy, Brian J; Lopez, Alejandro J; Menezes, Arnold H; Hitchon, Patrick W; Dahdaleh, Nader S

    2016-01-01

    Introduction: A rigid construct that employs an occipital plate and upper cervical screws and rods is the current standard treatment for craniovertebral junction (CVJ) instability. A rod is contoured to accommodate the occipitocervical angle. Fatigue failure has been associated these acute bends. Hinged rod systems have been developed to obviate intraoperative rod contouring. Object: The aim of this study is to determine the safety and efficacy of the hinged rod system in occipitocervical fusion. Materials and Methods: This study retrospectively evaluated 39 patients who underwent occipitocervical arthrodesis. Twenty patients were treated with hinged rods versus 19 with contoured rods. Clinical and radiographic data were compared and analyzed. Results: Preoperative and postoperative Nurick and Frankel scores were similar between both groups. The use of allograft, autograft or bone morphogenetic protein was similar in both groups. The average number of levels fused was 4.1 (±2.4) and 3.4 (±2) for hinged and contoured rods, respectively. The operative time, estimated blood loss, and length of stay were similar between both groups. The occiput to C2 angle was similarly maintained in both groups and all patients demonstrated no movement across the CVJ on flexion-extension X-rays during their last follow-up. The average follow-up for the hinged and contoured rod groups was 12.2 months and 15.9 months, respectively. Conclusion: Hinged rods provide a safe and effective alternative to contoured rods during occipitocervical arthrodesis.

  13. Comparison of hinged and contoured rods for occipitocervical arthrodesis in adults: A clinical study

    PubMed Central

    Abode-Iyamah, Kingsley O; Dlouhy, Brian J; Lopez, Alejandro J; Menezes, Arnold H; Hitchon, Patrick W; Dahdaleh, Nader S

    2016-01-01

    Introduction: A rigid construct that employs an occipital plate and upper cervical screws and rods is the current standard treatment for craniovertebral junction (CVJ) instability. A rod is contoured to accommodate the occipitocervical angle. Fatigue failure has been associated these acute bends. Hinged rod systems have been developed to obviate intraoperative rod contouring. Object: The aim of this study is to determine the safety and efficacy of the hinged rod system in occipitocervical fusion. Materials and Methods: This study retrospectively evaluated 39 patients who underwent occipitocervical arthrodesis. Twenty patients were treated with hinged rods versus 19 with contoured rods. Clinical and radiographic data were compared and analyzed. Results: Preoperative and postoperative Nurick and Frankel scores were similar between both groups. The use of allograft, autograft or bone morphogenetic protein was similar in both groups. The average number of levels fused was 4.1 (±2.4) and 3.4 (±2) for hinged and contoured rods, respectively. The operative time, estimated blood loss, and length of stay were similar between both groups. The occiput to C2 angle was similarly maintained in both groups and all patients demonstrated no movement across the CVJ on flexion-extension X-rays during their last follow-up. The average follow-up for the hinged and contoured rod groups was 12.2 months and 15.9 months, respectively. Conclusion: Hinged rods provide a safe and effective alternative to contoured rods during occipitocervical arthrodesis. PMID:27630479

  14. Protein function from its emergence to diversity in contemporary proteins

    NASA Astrophysics Data System (ADS)

    Goncearenco, Alexander; Berezovsky, Igor N.

    2015-07-01

    The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions—elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

  15. Protein function from its emergence to diversity in contemporary proteins.

    PubMed

    Goncearenco, Alexander; Berezovsky, Igor N

    2015-07-01

    The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions-elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

  16. Assigning protein functions by comparative genome analysis protein phylogenetic profiles

    DOEpatents

    Pellegrini, Matteo; Marcotte, Edward M.; Thompson, Michael J.; Eisenberg, David; Grothe, Robert; Yeates, Todd O.

    2003-05-13

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  17. Ribosomal proteins: functions beyond the ribosome

    PubMed Central

    Zhou, Xiang; Liao, Wen-Juan; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2015-01-01

    Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics. PMID:25735597

  18. Versatile hemidesmosomal linker proteins: structure and function.

    PubMed

    Chaudhari, Pratik R; Vaidya, Milind M

    2015-04-01

    Hemidesmosomes are anchoring junctions which connect basal epidermal cells to the extracellular matrix. In complex epithelia like skin, hemidesmosomes are composed of transmembrane proteins like α6β4 integrin, BP180, CD151 and cytoplasmic proteins like BPAG1e and plectin. BPAG1e and plectin are plakin family cytolinker proteins which anchor intermediate filament proteins i.e. keratins to the hemidesmosomal transmembrane proteins. Mutations in BPAG1e and plectin lead to severe skin blistering disorders. Recent reports indicate that these hemidesmosomal linker proteins play a role in various cellular processes like cell motility and cytoskeleton dynamics apart from their known anchoring function. In this review, we will discuss their role in structural and signaling functions.

  19. A review on protein functionalized carbon nanotubes.

    PubMed

    Nagaraju, Kathyayini; Reddy, Roopa; Reddy, Narendra

    2015-01-01

    Carbon nanotubes (CNTs) have been widely recognized and used for controlled drug delivery and in various other fields due to their unique properties and distinct advantages. Both single-walled carbon nanotubes (SWCNTs) and multiwalled (MWCNTs) carbon nanotubes are used and/or studied for potential applications in medical, energy, textile, composite, and other areas. Since CNTs are chemically inert and are insoluble in water or other organic solvents, they are functionalized or modified to carry payloads or interact with biological molecules. CNTs have been preferably functionalized with proteins because CNTs are predominantly used for medical applications such as delivery of drugs, DNA and genes, and also for biosensing. Extensive studies have been conducted to understand the interactions, cytotoxicity, and potential applications of protein functionalized CNTs but contradicting results have been published on the cytotoxicity of the functionalized CNTs. This paper provides a brief review of CNTs functionalized with proteins, methods used to functionalize the CNTs, and their potential applications. PMID:26660626

  20. A review on protein functionalized carbon nanotubes.

    PubMed

    Nagaraju, Kathyayini; Reddy, Roopa; Reddy, Narendra

    2015-12-18

    Carbon nanotubes (CNTs) have been widely recognized and used for controlled drug delivery and in various other fields due to their unique properties and distinct advantages. Both single-walled carbon nanotubes (SWCNTs) and multiwalled (MWCNTs) carbon nanotubes are used and/or studied for potential applications in medical, energy, textile, composite, and other areas. Since CNTs are chemically inert and are insoluble in water or other organic solvents, they are functionalized or modified to carry payloads or interact with biological molecules. CNTs have been preferably functionalized with proteins because CNTs are predominantly used for medical applications such as delivery of drugs, DNA and genes, and also for biosensing. Extensive studies have been conducted to understand the interactions, cytotoxicity, and potential applications of protein functionalized CNTs but contradicting results have been published on the cytotoxicity of the functionalized CNTs. This paper provides a brief review of CNTs functionalized with proteins, methods used to functionalize the CNTs, and their potential applications.

  1. Hinged, Magnetic Holder For Radiographic Film

    NASA Technical Reports Server (NTRS)

    Pierce, Darryl E.

    1989-01-01

    Hinged holder equipped with magnets enables positive, accurate, and repeatable placement and orientation of radiographic film at hidden and otherwise inaccessible location. Made from simple, readily available parts. Film and holder inserted in end of duct and pulled along by magnets on outside. Holder removed by reversing sequence of motions.

  2. Triangle-hinge models for unoriented membranes

    NASA Astrophysics Data System (ADS)

    Fukuma, Masafumi; Sugishita, Sotaro; Umeda, Naoya

    2016-07-01

    Triangle-hinge models [M. Fukuma, S. Sugishita, and N. Umeda, J. High Energy Phys. 1507, 088 (2015)] are introduced to describe worldvolume dynamics of membranes. The Feynman diagrams consist of triangles glued together along hinges and can be restricted to tetrahedral decompositions in a large-N limit. In this paper, after clarifying that all the tetrahedra resulting in the original models are orientable, we define a version of triangle-hinge models that can describe the dynamics of unoriented membranes. By regarding each triangle as representing a propagation of an open membrane of disk topology, we introduce a local worldvolume parity transformation which inverts the orientation of a triangle, and define unoriented triangle-hinge models by gauging the transformation. Unlike two-dimensional cases, this local transformation generally relates a manifold to a nonmanifold, but still is a well-defined manipulation among tetrahedral decompositions. We further show that matter fields can be introduced in the same way as in the original oriented models. In particular, the models will describe unoriented membranes in a target spacetime by taking matter fields to be the target space coordinates.

  3. 14 CFR 23.657 - Hinges.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... factor of safety of not less than 6.67 with respect to the ultimate bearing strength of the softest material used as a bearing. (b) For ball or roller bearing hinges, the approved rating of the bearing may... and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT...

  4. 14 CFR 25.657 - Hinges.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... safety of not less than 6.67 must be used with respect to the ultimate bearing strength of the softest material used as a bearing. (b) Hinges must have enough strength and rigidity for loads parallel to the... and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT...

  5. 14 CFR 25.657 - Hinges.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... safety of not less than 6.67 must be used with respect to the ultimate bearing strength of the softest material used as a bearing. (b) Hinges must have enough strength and rigidity for loads parallel to the... and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT...

  6. 14 CFR 23.657 - Hinges.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... factor of safety of not less than 6.67 with respect to the ultimate bearing strength of the softest material used as a bearing. (b) For ball or roller bearing hinges, the approved rating of the bearing may... and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT...

  7. Exploration of Multi-State Conformational Dynamics and Underlying Global Functional Landscape of Maltose Binding Protein

    PubMed Central

    Wang, Yong; Tang, Chun; Wang, Erkang; Wang, Jin

    2012-01-01

    An increasing number of biological machines have been revealed to have more than two macroscopic states. Quantifying the underlying multiple-basin functional landscape is essential for understanding their functions. However, the present models seem to be insufficient to describe such multiple-state systems. To meet this challenge, we have developed a coarse grained triple-basin structure-based model with implicit ligand. Based on our model, the constructed functional landscape is sufficiently sampled by the brute-force molecular dynamics simulation. We explored maltose-binding protein (MBP) which undergoes large-scale domain motion between open, apo-closed (partially closed) and holo-closed (fully closed) states responding to ligand binding. We revealed an underlying mechanism whereby major induced fit and minor population shift pathways co-exist by quantitative flux analysis. We found that the hinge regions play an important role in the functional dynamics as well as that increases in its flexibility promote population shifts. This finding provides a theoretical explanation of the mechanistic discrepancies in PBP protein family. We also found a functional “backtracking” behavior that favors conformational change. We further explored the underlying folding landscape in response to ligand binding. Consistent with earlier experimental findings, the presence of ligand increases the cooperativity and stability of MBP. This work provides the first study to explore the folding dynamics and functional dynamics under the same theoretical framework using our triple-basin functional model. PMID:22532792

  8. Flavin Redox Switching of Protein Functions

    PubMed Central

    Zhu, Weidong; Moxley, Michael A.

    2011-01-01

    Abstract Flavin cofactors impart remarkable catalytic diversity to enzymes, enabling them to participate in a broad array of biological processes. The properties of flavins also provide proteins with a versatile redox sensor that can be utilized for converting physiological signals such as cellular metabolism, light, and redox status into a unique functional output. The control of protein functions by the flavin redox state is important for transcriptional regulation, cell signaling pathways, and environmental adaptation. A significant number of proteins that have flavin redox switches are found in the Per-Arnt-Sim (PAS) domain family and include flavoproteins that act as photosensors and respond to changes in cellular redox conditions. Biochemical and structural studies of PAS domain flavoproteins have revealed key insights into how flavin redox changes are propagated to the surface of the protein and translated into a new functional output such as the binding of a target protein in a signaling pathway. Mechanistic details of proteins unrelated to the PAS domain are also emerging and provide novel examples of how the flavin redox state governs protein–membrane interactions in response to appropriate stimuli. Analysis of different flavin switch proteins reveals shared mechanistic themes for the regulation of protein structure and function by flavins. Antioxid. Redox Signal. 14, 1079–1091. PMID:21028987

  9. Integrating multiple networks for protein function prediction

    PubMed Central

    2015-01-01

    Background High throughput techniques produce multiple functional association networks. Integrating these networks can enhance the accuracy of protein function prediction. Many algorithms have been introduced to generate a composite network, which is obtained as a weighted sum of individual networks. The weight assigned to an individual network reflects its benefit towards the protein functional annotation inference. A classifier is then trained on the composite network for predicting protein functions. However, since these techniques model the optimization of the composite network and the prediction tasks as separate objectives, the resulting composite network is not necessarily optimal for the follow-up protein function prediction. Results We address this issue by modeling the optimization of the composite network and the prediction problems within a unified objective function. In particular, we use a kernel target alignment technique and the loss function of a network based classifier to jointly adjust the weights assigned to the individual networks. We show that the proposed method, called MNet, can achieve a performance that is superior (with respect to different evaluation criteria) to related techniques using the multiple networks of four example species (yeast, human, mouse, and fly) annotated with thousands (or hundreds) of GO terms. Conclusion MNet can effectively integrate multiple networks for protein function prediction and is robust to the input parameters. Supplementary data is available at https://sites.google.com/site/guoxian85/home/mnet. The Matlab code of MNet is available upon request. PMID:25707434

  10. Some functional properties of oilseed proteins.

    PubMed

    Khalil, M; Ragab, M; Hassanien, F R

    1985-01-01

    Oilseeds have potential food uses because of their high protein content. Besides, these proteins when added to a type of foods, supply desirable functional properties, such as whipping capacity and viscosity, emulsification and water and oil holding capacities. Rapeseed and soybean protein isolates were found to possess whipping capacity followed by those of sunflower, peanut, sesame, cottonseed and safflower. The addition of sugar improved the whipping properties of oilseed proteins. The whipping capacity of oilseed proteins decreased due to heating at 100 degrees C for time of 15 to 60 min. Soybean protein had the highest emulsifying capacity compared with the other oilseed proteins. The heated oilseed proteins had emulsification properties similar to or better than the control. Glandless cottonseed protein had high water and oil holding capacities. The water holding capacity of oilseed proteins decreased gradually as the duration of heating at 100 degrees C was increased. On the other hand the heated oilseed proteins had oil holding capacities similar to or better than unheated proteins. PMID:4000248

  11. Measuring the functional sequence complexity of proteins

    PubMed Central

    Durston, Kirk K; Chiu, David KY; Abel, David L; Trevors, Jack T

    2007-01-01

    Background Abel and Trevors have delineated three aspects of sequence complexity, Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC) observed in biosequences such as proteins. In this paper, we provide a method to measure functional sequence complexity. Methods and Results We have extended Shannon uncertainty by incorporating the data variable with a functionality variable. The resulting measured unit, which we call Functional bit (Fit), is calculated from the sequence data jointly with the defined functionality variable. To demonstrate the relevance to functional bioinformatics, a method to measure functional sequence complexity was developed and applied to 35 protein families. Considerations were made in determining how the measure can be used to correlate functionality when relating to the whole molecule and sub-molecule. In the experiment, we show that when the proposed measure is applied to the aligned protein sequences of ubiquitin, 6 of the 7 highest value sites correlate with the binding domain. Conclusion For future extensions, measures of functional bioinformatics may provide a means to evaluate potential evolving pathways from effects such as mutations, as well as analyzing the internal structural and functional relationships within the 3-D structure of proteins. PMID:18062814

  12. Activation of complement by an IgG molecule without a genetic hinge.

    PubMed

    Brekke, O H; Michaelsen, T E; Sandin, R; Sandlie, I

    1993-06-17

    The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

  13. Evolution of Ftz protein function in insects.

    PubMed

    Alonso, C R; Maxton-Kuechenmeister, J; Akam, M

    2001-09-18

    The Drosophila gene fushi tarazu (ftz) encodes a homeodomain-containing transcriptional regulator (Ftz) required at several stages during development. Drosophila melanogaster ftz (Dm-ftz) is first expressed in seven stripes defining alternate parasegments of the embryo--a "pair-rule" segmentation function [1, 2]. It is then expressed in specific neural precursor cells in the central nervous system and finally in the developing hindgut [3]. An Orthopteran ortholog of ftz (Sg-ftz, formally Dax) has been isolated from the grasshopper Schistocerca gregaria [4]. The pattern of Sg-ftz expression in Schistocerca embryos suggests that some developmental roles of the ftz gene are likely to be conserved between these two species (e.g., CNS functions) while others may have diverged (e.g., segmentation functions). To test whether the function of the Ftz protein itself differs between these two species, here we compare the functions of Sg-Ftz and Dm-Ftz proteins by expressing both in Drosophila embryos. Sg-ftz mimics only poorly several segmentation roles of Dm-ftz (engrailed activation, wingless repression, and embryonic cuticle transformation). However, the two proteins are similarly active in the rescue of a CNS-specific ftz mutant. These findings argue that this ftz CNS function is mediated by conserved parts of the protein, while efficient pair-rule function requires sequences present specifically in the Drosophila protein. PMID:11566109

  14. Genetically modified proteins: functional improvement and chimeragenesis

    PubMed Central

    Balabanova, Larissa; Golotin, Vasily; Podvolotskaya, Anna; Rasskazov, Valery

    2015-01-01

    This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit. PMID:26211369

  15. Profiling protein function with small molecule microarrays

    PubMed Central

    Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

    2002-01-01

    The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

  16. Thin film solar cell inflatable ultraviolet rigidizable deployment hinge

    NASA Technical Reports Server (NTRS)

    Simburger, Edward J. (Inventor); Matsumoto, James H. (Inventor); Giants, Thomas W. (Inventor); Garcia, III, Alec (Inventor); Perry, Alan R. (Inventor); Rawal, Suraj (Inventor); Marshall, Craig H. (Inventor); Lin, John K. H. (Inventor); Day, Jonathan Robert (Inventor); Kerslake, Thomas W. (Inventor)

    2010-01-01

    A flexible inflatable hinge includes curable resin for rigidly positioning panels of solar cells about the hinge in which wrap around contacts and flex circuits are disposed for routing power from the solar cells to the power bus further used for grounding the hinge. An indium tin oxide and magnesium fluoride coating is used to prevent static discharge while being transparent to ultraviolet light that cures the embedded resin after deployment for rigidizing the inflatable hinge.

  17. Functional annotation of hypothetical proteins - A review.

    PubMed

    Sivashankari, Selvarajan; Shanmughavel, Piramanayagam

    2006-12-29

    The complete human genome sequences in the public database provide ways to understand the blue print of life. As of June 29, 2006, 27 archaeal, 326 bacterial and 21 eukaryotes is complete genomes are available and the sequencing for 316 bacterial, 24 archaeal, 126 eukaryotic genomes are in progress. The traditional biochemical/molecular experiments can assign accurate functions for genes in these genomes. However, the process is time-consuming and costly. Despite several efforts, only 50-60 % of genes have been annotated in most completely sequenced genomes. Automated genome sequence analysis and annotation may provide ways to understand genomes. Thus, determination of protein function is one of the challenging problems of the post-genome era. This demands bioinformatics to predict functions of un-annotated protein sequences by developing efficient tools. Here, we discuss some of the recent and popular approaches developed in Bioinformatics to predict functions for hypothetical proteins.

  18. Template-based prediction of protein function.

    PubMed

    Petrey, Donald; Chen, T Scott; Deng, Lei; Garzon, Jose Ignacio; Hwang, Howook; Lasso, Gorka; Lee, Hunjoong; Silkov, Antonina; Honig, Barry

    2015-06-01

    We discuss recent approaches for structure-based protein function annotation. We focus on template-based methods where the function of a query protein is deduced from that of a template for which both the structure and function are known. We describe the different ways of identifying a template. These are typically based on sequence analysis but new methods based on purely structural similarity are also being developed that allow function annotation based on structural relationships that cannot be recognized by sequence. The growing number of available structures of known function, improved homology modeling techniques and new developments in the use of structure allow template-based methods to be applied on a proteome-wide scale and in many different biological contexts. This progress significantly expands the range of applicability of structural information in function annotation to a level that previously was only achievable by sequence comparison.

  19. Quantitative assessment of protein function prediction programs.

    PubMed

    Rodrigues, B N; Steffens, M B R; Raittz, R T; Santos-Weiss, I C R; Marchaukoski, J N

    2015-12-21

    Fast prediction of protein function is essential for high-throughput sequencing analysis. Bioinformatic resources provide cheaper and faster techniques for function prediction and have helped to accelerate the process of protein sequence characterization. In this study, we assessed protein function prediction programs that accept amino acid sequences as input. We analyzed the classification, equality, and similarity between programs, and, additionally, compared program performance. The following programs were selected for our assessment: Blast2GO, InterProScan, PANTHER, Pfam, and ScanProsite. This selection was based on the high number of citations (over 500), fully automatic analysis, and the possibility of returning a single best classification per sequence. We tested these programs using 12 gold standard datasets from four different sources. The gold standard classification of the databases was based on expert analysis, the Protein Data Bank, or the Structure-Function Linkage Database. We found that the miss rate among the programs is globally over 50%. Furthermore, we observed little overlap in the correct predictions from each program. Therefore, a combination of multiple types of sources and methods, including experimental data, protein-protein interaction, and data mining, may be the best way to generate more reliable predictions and decrease the miss rate.

  20. Evolution-Based Functional Decomposition of Proteins.

    PubMed

    Rivoire, Olivier; Reynolds, Kimberly A; Ranganathan, Rama

    2016-06-01

    The essential biological properties of proteins-folding, biochemical activities, and the capacity to adapt-arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment-a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation. PMID:27254668

  1. Quantitative assessment of protein function prediction programs.

    PubMed

    Rodrigues, B N; Steffens, M B R; Raittz, R T; Santos-Weiss, I C R; Marchaukoski, J N

    2015-01-01

    Fast prediction of protein function is essential for high-throughput sequencing analysis. Bioinformatic resources provide cheaper and faster techniques for function prediction and have helped to accelerate the process of protein sequence characterization. In this study, we assessed protein function prediction programs that accept amino acid sequences as input. We analyzed the classification, equality, and similarity between programs, and, additionally, compared program performance. The following programs were selected for our assessment: Blast2GO, InterProScan, PANTHER, Pfam, and ScanProsite. This selection was based on the high number of citations (over 500), fully automatic analysis, and the possibility of returning a single best classification per sequence. We tested these programs using 12 gold standard datasets from four different sources. The gold standard classification of the databases was based on expert analysis, the Protein Data Bank, or the Structure-Function Linkage Database. We found that the miss rate among the programs is globally over 50%. Furthermore, we observed little overlap in the correct predictions from each program. Therefore, a combination of multiple types of sources and methods, including experimental data, protein-protein interaction, and data mining, may be the best way to generate more reliable predictions and decrease the miss rate. PMID:26782400

  2. Calreticulin: one protein, one gene, many functions.

    PubMed Central

    Michalak, M; Corbett, E F; Mesaeli, N; Nakamura, K; Opas, M

    1999-01-01

    The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology. PMID:10567207

  3. Investigating neuronal function with optically controllable proteins

    PubMed Central

    Zhou, Xin X.; Pan, Michael; Lin, Michael Z.

    2015-01-01

    In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain. PMID:26257603

  4. Protein-protein interaction network-based detection of functionally similar proteins within species.

    PubMed

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.

  5. Evolution-Based Functional Decomposition of Proteins

    PubMed Central

    Rivoire, Olivier; Reynolds, Kimberly A.; Ranganathan, Rama

    2016-01-01

    The essential biological properties of proteins—folding, biochemical activities, and the capacity to adapt—arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment—a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation. PMID:27254668

  6. Evidence on How a Conserved Glycine in the Hinge Region of HapR Regulates Its DNA Binding Ability: LESSONS FROM A NATURAL VARIANT.

    SciTech Connect

    M Dongre; N Singh; C Dureja; N Peddada; A Solanki; F Ashish; S Raychaudhuri

    2011-12-31

    HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapRV2) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapRV2 to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapRV2 (HapRV2G), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapRV2 and HapRV2G proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a 'Y' shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

  7. Protein function prediction using neighbor relativity in protein-protein interaction network.

    PubMed

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network.

  8. Elbow- and hinge-bending motions of IgG: Dielectric response and dynamic feature.

    PubMed

    Hayashi, Yoshihito; Yagihara, Shin

    2016-09-01

    Immunoglobulin G (IgG) is a Y-shaped globular protein consisting of two Fab segments connecting to an Fc segment with a flexible hinge region, in which the Fab segments show secondary flexibility at an "elbow" region. In the present work, the hinge-bending and elbow-bending motions of aqueous solutions of IgG by microwave dielectric measurements below the freezing point of bulk water was observed. The presence of unfreezable water around the macromolecules reduced the effects of steric hindrance normally generated by ice and enabled the intramolecular motions of IgG. At the same time, the overall IgG molecule rotation was restricted by ice. Papain digestion and reduction of the disulfide linkage at the hinge region was used to generate Fab and Fc fragments. In solutions of these fragments, the dielectric relaxation process of the hinge-bending motion was absent, although the elbow-bending motion remained. Three relaxation processes were observed for papain-digested IgG. The high, middle, and low frequency processes were attributed to unfrozen water, local peptide motions cooperating with bound water, and the elbow-bending motion, respectively. In the case of the intact IgG, an additional relaxation process due to the hinge-bending motion was observed at frequencies lower than that of the elbow-bending motion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 626-632, 2016. PMID:27018805

  9. Proteins with Novel Structure, Function and Dynamics

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew

    2014-01-01

    Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

  10. Functions of TET Proteins in Hematopoietic Transformation.

    PubMed

    Han, Jae-A; An, Jungeun; Ko, Myunggon

    2015-11-01

    DNA methylation is a well-characterized epigenetic modification that plays central roles in mammalian development, genomic imprinting, X-chromosome inactivation and silencing of retrotransposon elements. Aberrant DNA methylation pattern is a characteristic feature of cancers and associated with abnormal expression of oncogenes, tumor suppressor genes or repair genes. Ten-eleven-translocation (TET) proteins are recently characterized dioxygenases that catalyze progressive oxidation of 5-methylcytosine to produce 5-hydroxymethylcytosine and further oxidized derivatives. These oxidized methylcytosines not only potentiate DNA demethylation but also behave as independent epigenetic modifications per se. The expression or activity of TET proteins and DNA hydroxymethylation are highly dysregulated in a wide range of cancers including hematologic and non-hematologic malignancies, and accumulating evidence points TET proteins as a novel tumor suppressor in cancers. Here we review DNA demethylation-dependent and -independent functions of TET proteins. We also describe diverse TET loss-of-function mutations that are recurrently found in myeloid and lymphoid malignancies and their potential roles in hematopoietic transformation. We discuss consequences of the deficiency of individual Tet genes and potential compensation between different Tet members in mice. Possible mechanisms underlying facilitated oncogenic transformation of TET-deficient hematopoietic cells are also described. Lastly, we address non-mutational mechanisms that lead to suppression or inactivation of TET proteins in cancers. Strategies to restore normal 5mC oxidation status in cancers by targeting TET proteins may provide new avenues to expedite the development of promising anti-cancer agents.

  11. The lipocalin protein family: structure and function.

    PubMed Central

    Flower, D R

    1996-01-01

    The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds. PMID:8761444

  12. Hierarchical Ensemble Methods for Protein Function Prediction

    PubMed Central

    2014-01-01

    Protein function prediction is a complex multiclass multilabel classification problem, characterized by multiple issues such as the incompleteness of the available annotations, the integration of multiple sources of high dimensional biomolecular data, the unbalance of several functional classes, and the difficulty of univocally determining negative examples. Moreover, the hierarchical relationships between functional classes that characterize both the Gene Ontology and FunCat taxonomies motivate the development of hierarchy-aware prediction methods that showed significantly better performances than hierarchical-unaware “flat” prediction methods. In this paper, we provide a comprehensive review of hierarchical methods for protein function prediction based on ensembles of learning machines. According to this general approach, a separate learning machine is trained to learn a specific functional term and then the resulting predictions are assembled in a “consensus” ensemble decision, taking into account the hierarchical relationships between classes. The main hierarchical ensemble methods proposed in the literature are discussed in the context of existing computational methods for protein function prediction, highlighting their characteristics, advantages, and limitations. Open problems of this exciting research area of computational biology are finally considered, outlining novel perspectives for future research. PMID:25937954

  13. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

    Rubinstein, Rotem; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.; Fiser, Andras

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  14. Modification of sorghum proteins for enhanced functionality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum is the third most widely produced crop in the United States (U.S.) and fifth in the world during fiscal year 2006/07(USDA-FAS, 2007). The use of sorghum in foods faces functional and nutritional constraints due, mainly, to the rigidity of the protein bodies. The disruption and modificatio...

  15. Functional prediction of hypothetical proteins in human adenoviruses.

    PubMed

    Dorden, Shane; Mahadevan, Padmanabhan

    2015-01-01

    Assigning functional information to hypothetical proteins in virus genomes is crucial for gaining insight into their proteomes. Human adenoviruses are medium sized viruses that cause a range of diseases. Their genomes possess proteins with uncharacterized function known as hypothetical proteins. Using a wide range of protein function prediction servers, functional information was obtained about these hypothetical proteins. A comparison of functional information obtained from these servers revealed that some of them produced functional information, while others provided little functional information about these human adenovirus hypothetical proteins. The PFP, ESG, PSIPRED, 3d2GO, and ProtFun servers produced the most functional information regarding these hypothetical proteins. PMID:26664031

  16. Insect Seminal Fluid Proteins: Identification and Function

    PubMed Central

    Avila, Frank W.; Sirot, Laura K.; LaFlamme, Brooke A.; Rubinstein, C. Dustin; Wolfner, Mariana F.

    2014-01-01

    Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects. PMID:20868282

  17. Ice-Binding Proteins and Their Function.

    PubMed

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  18. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria.

    PubMed

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-10-22

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and "interologs" in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria.

  19. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria

    PubMed Central

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-01-01

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and “interologs” in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

  20. Thermoelectric generator with hinged assembly for fins

    DOEpatents

    Purdy, David L.; Shapiro, Zalman M.; Hursen, Thomas F.; Maurer, Gerould W.

    1976-11-02

    A cylindrical casing has a central shielded capsule of radioisotope fuel. A plurality of thermonuclear modules are axially arranged with their hot junctions resiliently pressed toward the shield and with their cold junctions adjacent a transition member having fins radiating heat to the environment. For each module, the assembly of transition member and fins is hinged to the casing for swinging to permit access to and removal of such module. A ceramic plate having gold layers on opposite faces prevents diffusion bonding of the hot junction to the shield.

  1. ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding

    PubMed Central

    Akai, Yuko; Kanai, Ryuta; Nakazawa, Norihiko; Ebe, Masahiro; Toyoshima, Chikashi; Yanagida, Mitsuhiro

    2014-01-01

    Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA. PMID:25520186

  2. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  3. Functional Differences in Yeast Protein Disulfide Isomerases

    PubMed Central

    Nørgaard, Per; Westphal, Vibeke; Tachibana, Christine; Alsøe, Lene; Holst, Bjørn; Winther, Jakob R.

    2001-01-01

    PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains. PMID:11157982

  4. YB-1 protein: functions and regulation.

    PubMed

    Lyabin, Dmitry N; Eliseeva, Irina A; Ovchinnikov, Lev P

    2014-01-01

    The Y-box binding protein 1 (YB-1, YBX1) is a member of the family of DNA- and RNA-binding proteins with an evolutionarily ancient and conserved cold shock domain. It falls into a group of intrinsically disordered proteins that do not follow the classical rule 'one protein-one function' but introduce a novel principle stating that a disordered structure suggests many functions. YB-1 participates in a wide variety of DNA/RNA-dependent events, including DNA reparation, pre-mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. At the cell level, the multiple activities of YB-1 are manifested as its involvement in cell proliferation and differentiation, stress response, and malignant cell transformation. WIREs RNA 2014, 5:95-110. doi: 10.1002/wrna.1200 CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

  5. Predicting Protein Function Using Multiple Kernels.

    PubMed

    Yu, Guoxian; Rangwala, Huzefa; Domeniconi, Carlotta; Zhang, Guoji; Zhang, Zili

    2015-01-01

    High-throughput experimental techniques provide a wide variety of heterogeneous proteomic data sources. To exploit the information spread across multiple sources for protein function prediction, these data sources are transformed into kernels and then integrated into a composite kernel. Several methods first optimize the weights on these kernels to produce a composite kernel, and then train a classifier on the composite kernel. As such, these approaches result in an optimal composite kernel, but not necessarily in an optimal classifier. On the other hand, some approaches optimize the loss of binary classifiers and learn weights for the different kernels iteratively. For multi-class or multi-label data, these methods have to solve the problem of optimizing weights on these kernels for each of the labels, which are computationally expensive and ignore the correlation among labels. In this paper, we propose a method called Predicting Protein Function using Multiple Kernels (ProMK). ProMK iteratively optimizes the phases of learning optimal weights and reduces the empirical loss of multi-label classifier for each of the labels simultaneously. ProMK can integrate kernels selectively and downgrade the weights on noisy kernels. We investigate the performance of ProMK on several publicly available protein function prediction benchmarks and synthetic datasets. We show that the proposed approach performs better than previously proposed protein function prediction approaches that integrate multiple data sources and multi-label multiple kernel learning methods. The codes of our proposed method are available at https://sites.google.com/site/guoxian85/promk.

  6. Heterogeneity in Retroviral Nucleocapsid Protein Function

    NASA Astrophysics Data System (ADS)

    Landes, Christy

    2009-03-01

    Time-resolved single-molecule fluorescence spectroscopy was used to study the human T-cell lymphotropic virus type 1 (HTLV-1) nucleocapsid protein (NC) chaperone activity as compared to that of the HIV-1 NC protein. HTLV-1 NC contains two zinc fingers with each having a CCHC binding motif similar to HIV-1 NC. HIV-1 NC is required for recognition and packaging of the viral RNA and is also a nucleic acid chaperone protein that facilitates nucleic acid restructuring during reverse transcription. Because of similarities in structures between the two retroviruses, we have used single-molecule fluorescence energy transfer to investigate the chaperoning activity of HTLV-1 NC protein. The results indicate that HTLV-1 NC protein induces structural changes by opening the transactivation response (TAR)-DNA hairpin to an even greater extent than HIV-1 NC. However, unlike HIV-1 NC, HTLV-1 NC does not chaperone the strand-transfer reaction involving TAR-DNA. These results suggest that despite its effective destabilization capability, HTLV-1 NC is not as effective at overall chaperone function as is its HIV-1 counterpart.

  7. Recognition of galactose-deficient O-glycans in the hinge region of IgA1 by N-acetylgalactosamine-specific snail lectins: a comparative binding study.

    PubMed

    Gomes, Michelle M; Suzuki, Hitoshi; Brooks, Monica T; Tomana, Milan; Moldoveanu, Zina; Mestecky, Jiri; Julian, Bruce A; Novak, Jan; Herr, Andrew B

    2010-07-13

    Aberrancies in IgA1 glycosylation have been linked to the pathogenesis of IgA nephropathy (IgAN), a kidney disease characterized by deposits of IgA1-containing immune complexes in the glomerular mesangium. IgA1 from IgAN patients is characterized by the presence of galactose (Gal)-deficient O-glycans in the hinge region that can act as epitopes for anti-glycan IgG or IgA1 antibodies. The resulting circulating immune complexes are trapped in the glomerular mesangium of the kidney where they trigger localized inflammatory responses by activating mesangial cells. Certain lectins recognize the terminal N-acetylgalactosamine (GalNAc)-containing O-glycans on Gal-deficient IgA1 and can be potentially used as diagnostic tools. To improve our understanding of GalNAc recognition by these lectins, we have conducted binding studies to assess the interaction of Helix aspersa agglutinin (HAA) and Helix pomatia agglutinin (HPA) with Gal-deficient IgA1. Surface plasmon resonance spectroscopy revealed that both HAA and HPA bind to a Gal-deficient synthetic hinge region glycopeptide (HR-GalNAc) as well as various aberrantly glycosylated IgA1 myeloma proteins. Despite having six binding sites, both HAA and HPA bind IgA1 in a functionally bivalent manner, with the apparent affinity for IgA1 related to the number of exposed GalNAc groups in the IgA1 hinge. Finally, HAA and HPA were shown to discriminate very effectively between the IgA1 secreted by cell lines derived from peripheral blood cells of patients with IgAN and that from cells of healthy controls. These studies provide insight into lectin recognition of the Gal-deficient IgA1 hinge region and lay the groundwork for the development of reliable diagnostic tools for IgAN.

  8. Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge.

    PubMed

    Norderhaug, L; Brekke, O H; Bremnes, B; Sandin, R; Aase, A; Michaelsen, T E; Sandlie, I

    1991-10-01

    We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.

  9. roof truss detail, historic strap hinge detail Chopawamsic Recreational ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    roof truss detail, historic strap hinge detail - Chopawamsic Recreational Demonstration Area - Cabin Camp 1, Main Arts and Crafts Lodge, Prince William Forest Park, Triangle, Prince William County, VA

  10. Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition.

    PubMed

    Uchiyama, Susumu; Kawahara, Kazuki; Hosokawa, Yuki; Fukakusa, Shunsuke; Oki, Hiroya; Nakamura, Shota; Kojima, Yukiko; Noda, Masanori; Takino, Rie; Miyahara, Yuya; Maruno, Takahiro; Kobayashi, Yuji; Ohkubo, Tadayasu; Fukui, Kiichi

    2015-12-01

    Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.

  11. Structural and Inhibitory Effects of Hinge Loop Mutagenesis in Serpin-2 from the Malaria Vector Anopheles gambiae*

    PubMed Central

    Zhang, Xin; Meekins, David A.; An, Chunju; Zolkiewski, Michal; Battaile, Kevin P.; Kanost, Michael R.; Lovell, Scott; Michel, Kristin

    2015-01-01

    Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9), which functions in a serine proteinase cascade culminating in the activation of prophenoloxidase and melanization. Silencing of SRPN2 in A. gambiae results in spontaneous melanization and decreased life span and is therefore a promising target for vector control. The previously determined structure of SRPN2 revealed a partial insertion of the hinge region of the reactive center loop (RCL) into β sheet A. This partial hinge insertion participates in heparin-linked activation in other serpins, notably antithrombin III. SRPN2 does not contain a heparin binding site, and any possible mechanistic function of the hinge insertion was previously unknown. To investigate the function of the SRPN2 hinge insertion, we developed three SRPN2 variants in which the hinge regions are either constitutively expelled or inserted and analyzed their structure, thermostability, and inhibitory activity. We determined that constitutive hinge expulsion resulted in a 2.7-fold increase in the rate of CLIPB9Xa inhibition, which is significantly lower than previous observations of allosteric serpin activation. Furthermore, we determined that stable insertion of the hinge region did not appreciably decrease the accessibility of the RCL to CLIPB9. Together, these results indicate that the partial hinge insertion in SRPN2 does not participate in the allosteric activation observed in other serpins and instead represents a molecular trade-off between RCL accessibility and efficient formation of an inhibitory complex with the cognate proteinase. PMID:25525260

  12. The Amyloid Precursor Protein Controls PIKfyve Function

    PubMed Central

    Balklava, Zita; Niehage, Christian; Currinn, Heather; Mellor, Laura; Guscott, Benjamin; Poulin, Gino; Hoflack, Bernard; Wassmer, Thomas

    2015-01-01

    While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease. PMID:26125944

  13. The Amyloid Precursor Protein Controls PIKfyve Function.

    PubMed

    Balklava, Zita; Niehage, Christian; Currinn, Heather; Mellor, Laura; Guscott, Benjamin; Poulin, Gino; Hoflack, Bernard; Wassmer, Thomas

    2015-01-01

    While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease. PMID:26125944

  14. Mechanically Biased, Hinged Pairs of Piezoelectric Benders

    NASA Technical Reports Server (NTRS)

    Sager, Frank E.

    2005-01-01

    The upper part of the figure depicts an actuator that comprises two mechanically biased piezoelectric benders hinged together at their ends and equipped with tabs at their mid-length points for attachment to the relatively moving objects that are to be actuated. In the example of the figure, the attachment tabs are labeled to indicate that the actuator is used to drive a pump piston relative to a base plate. Actuators of this type could be used to drive low-power, small-volume pumps in consumer, medical, and aerospace applications, and to generate and measure linear displacements in such robotic applications as teleoperation and tactile feedback. Each bender is a bimorph a unitary plate that comprises an upper and a lower piezoelectric layer plus electrode layers. Benders may also be made of several layers arranged to produce the same effect at the lower operating voltages. As stated above, each bender is mechanically biased; it is fabricated to have a small permanent curvature (the bias curvature) in the absence of applied voltage. As on other bimorphs, the electrical connections on each bender are arranged so that an applied voltage of suitable polarity causes the upper layer to expand and the lower layer to contract. In this case, the net effect of applying the voltage is that the plate becomes more concave as viewed from below. Conversely, an applied voltage of the opposite polarity causes the plate to become less concave as viewed from below. The benders in a hinged pair are oriented with their bias curvatures concave inward, so that there is a bias distance between the attachment tabs. The two benders are connected electrically in parallel, with their connection polarities chosen so that an applied voltage of one polarity causes both benders to become more convex inward (more bent), while an applied voltage of the opposite polarity causes both benders to become less convex inward (less bent). An increase or decrease in bend is accompanied by an increase or

  15. [Location and functions of secretagogin protein].

    PubMed

    Liu, Qin; Lai, Maode

    2016-01-01

    Secretagogin (SCGN) is a novel member of EF-hand Ca2+-binding proteins, which was identified in islet β cells by Wagner. SCGN is a six EF-hand Ca2+-binding protein, primarily expressed on the neuroendocrine axis and the central nervous system. The protein has abundant biological functions. A certain concentration of calcium ion can lead to conformation change of SCGN, resulting in the change of intracellular signal transduction. Preliminary studies showed that SCGN would be used to treat stress reaction, such as mental illness (depression), burns or post-traumatic stress disorder and chronic stress reaction caused by pain. In Alzheimer's disease, the expression of SCGN in the hippocampus can boycott neurodegeneration. In neuroendocrine tumors, SCGN presents a good consistency with neuroendocrine markers such as CgA, Syn, and NSE, with a higher overall sensitivity and specificity. In addition, SCGN is released into serum after neural damage in cerebral ischemic diseases, suggesting that SCGN can be used as a marker for brain trauma. In this article, we review the recent research progress of secretagogin, focus on its distribution and functions in various tumorous diseases and non-tumorous diseases, such as Alzheimer's disease. PMID:27045242

  16. Nanostructured functional films from engineered repeat proteins

    PubMed Central

    Grove, Tijana Z.; Regan, Lynne; Cortajarena, Aitziber L.

    2013-01-01

    Fundamental advances in biotechnology, medicine, environment, electronics and energy require methods for precise control of spatial organization at the nanoscale. Assemblies that rely on highly specific biomolecular interactions are an attractive approach to form materials that display novel and useful properties. Here, we report on assembly of films from the designed, rod-shaped, superhelical, consensus tetratricopeptide repeat protein (CTPR). We have designed three peptide-binding sites into the 18 repeat CTPR to allow for further specific and non-covalent functionalization of films through binding of fluorescein labelled peptides. The fluorescence signal from the peptide ligand bound to the protein in the solid film is anisotropic, demonstrating that CTPR films can impose order on otherwise isotropic moieties. Circular dichroism measurements show that the individual protein molecules retain their secondary structure in the film, and X-ray scattering, birefringence and atomic force microscopy experiments confirm macroscopic alignment of CTPR molecules within the film. This work opens the door to the generation of innovative biomaterials with tailored structure and function. PMID:23594813

  17. [Functions of prion protein PrPc].

    PubMed

    Cazaubon, Sylvie; Viegas, Pedro; Couraud, Pierre-Olivier

    2007-01-01

    It is now well established that both normal and pathological (or scrapie) isoforms of prion protein, PrPc and PrPsc respectively, are involved in the development and progression of various forms of neurodegenerative diseases, including scrapie in sheep, bovine spongiform encephalopathy (or "mad cow disease") and Creutzfeldt-Jakob disease in human, collectively known as prion diseases. The protein PrPc is highly expressed in the central nervous system in neurons and glial cells, and also present in non-brain cells, such as immune cells or epithelial and endothelial cells. Identification of the physiological functions of PrPc in these different cell types thus appears crucial for understanding the progression of prion diseases. Recent studies highlighted several major roles for PrPc that may be considered in two major domains : (1) cell survival (protection against oxidative stress and apoptosis) and (2) cell adhesion. In association with cell adhesion, distinct functions of PrPc were observed, depending on cell types : neuronal differentiation, epithelial and endothelial barrier integrity, transendothelial migration of monocytes, T cell activation. These observations suggest that PrPc functions may be particularly relevant to cellular stress, as well as inflammatory or infectious situations. PMID:17875293

  18. Drosophila mechanotransduction--linking proteins and functions.

    PubMed

    Albert, Jörg T; Nadrowski, Björn; Göpfert, Martin C

    2007-01-01

    The sensation of touch, gravity, and sound all rely on dedicated ion channels that transduce mechanical stimulus forces into electrical signals. The functional workings and molecular identities of these mechanotransducer channels are little understood. Recent work shows that the mechanotransducers for fly and vertebrate hearing share equivalent gating mechanisms, whereby this mechanism can be probed non-invasively in the mechanics of the Drosophila ear. Here, we describe how this mechanics can be used to evaluate the roles of identified proteins in the process of mechanosensation and, specifically, their contributions to mechanotransduction. PMID:18820433

  19. Functions and possible provenance of primordial proteins.

    PubMed

    Sommer, Andrei P; Miyake, Norimune; Wickramasinghe, N Chandra; Narlikar, Jayant V; Al-Mufti, Shirwan

    2004-01-01

    Nanobacteria or living nanovesicles are of great interest to the scientific community because of their dual nature: on the one hand, they appear as primal biosystems originating life; on the other hand, they can cause severe diseases. Their survival as well as their pathogenic potential is apparently linked to a self-synthesized protein-based slime, rich in calcium and phosphate (when available). Here, we provide challenging evidence for the occurrence of nanobacteria in the stratosphere, reflecting a possibly primordial provenance of the slime. An analysis of the slime's biological functions may lead to novel strategies suitable to block adhesion modalities in modern bacterial populations. PMID:15595742

  20. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  1. Vibration control of flexible beams using an active hinge

    NASA Technical Reports Server (NTRS)

    Cudney, H. H., Jr.; Inman, D. J.; Horner, G. C.

    1985-01-01

    The use of an active hinge to attenuate the transverse vibrations of a flexible beam is examined. A slender aluminum beam is suspended vertically, cantilevered at the top. An active hinge is placed at the node of the second vibration mode. The active hinge consists of a torque motor, strain gauge, and tachometer. A control law is implemented using both beam-bending strain and the relative angular velocity measured at this hinge, thereby configuring the hinge to act as an active damper. Results from implementing this control law show little improvement in the first mode damping ratio, 130 percent increase in the second mode damping ratio, and 180 percent increase in the third mode damping ratio. The merits of using a motor with a gearbox are discussed.

  2. Multiple functions of microsomal triglyceride transfer protein

    PubMed Central

    2012-01-01

    Microsomal triglyceride transfer protein (MTP) was first identified as a major cellular protein capable of transferring neutral lipids between membrane vesicles. Its role as an essential chaperone for the biosynthesis of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins was established after the realization that abetalipoproteinemia patients carry mutations in the MTTP gene resulting in the loss of its lipid transfer activity. Now it is known that it also plays a role in the biosynthesis of CD1, glycolipid presenting molecules, as well as in the regulation of cholesterol ester biosynthesis. In this review, we will provide a historical perspective about the identification, purification and characterization of MTP, describe methods used to measure its lipid transfer activity, and discuss tissue expression and function. Finally, we will review the role MTP plays in the assembly of apoB-lipoprotein, the regulation of cholesterol ester synthesis, biosynthesis of CD1 proteins and propagation of hepatitis C virus. We will also provide a brief overview about the clinical potentials of MTP inhibition. PMID:22353470

  3. Effects of ozone on functional properties of proteins.

    PubMed

    Uzun, Hicran; Ibanoglu, Esra; Catal, Hatice; Ibanoglu, Senol

    2012-09-15

    The present study investigates whether the ozone treatment could be an alternative to improve some functional properties of proteins. Ozone treatment was applied on whey protein isolate and egg white proteins which have been extensively used in food products to improve textural, functional and sensory attributes. Ozone treatment of proteins was performed either in aqueous solutions or as gas ozonation of pure protein powders. Foam formation and foam stability of proteins were enhanced extensively. The solubility of proteins were reduced as influenced from the aqueous and gas ozonation medium. The reduction was more pronounced in egg white proteins. Ozone treatment affected emulsion activity of whey protein isolate negatively and reduced the emulsion stability.

  4. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    NASA Astrophysics Data System (ADS)

    Kim, Young Eun; Kim, Yu-Na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-05-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

  5. Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Pellegrini, Matteo; Thompson, Michael J.; Yeates, Todd O.

    2002-10-15

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  6. Characterization of the functional properties of carob germ proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins from the carob germ were identified as having gluten-like proteins in 1935. While some biochemical characterization of carob germ proteins and their functionality has been carried out, relatively little has been done when compared to proteins such as gluten. Carob germ proteins were separ...

  7. A novel genetic mechanism regulates dorsolateral hinge-point formation during zebrafish cranial neurulation.

    PubMed

    Nyholm, Molly K; Abdelilah-Seyfried, Salim; Grinblat, Yevgenya

    2009-06-15

    During neurulation, vertebrate embryos form a neural tube (NT), the rudiment of the central nervous system. In mammals and birds, a key step in cranial NT morphogenesis is dorsolateral hinge-point (DLHP) bending, which requires an apical actomyosin network. The mechanism of DLHP formation is poorly understood, although several essential genes have been identified, among them Zic2, which encodes a zinc-finger transcription factor. We found that DLHP formation in the zebrafish midbrain also requires actomyosin and Zic function. Given this conservation, we used the zebrafish to study how genes encoding Zic proteins regulate DLHP formation. We demonstrate that the ventral zic2a expression border predicts DLHP position. Using morpholino (MO) knockdown, we show zic2a and zic5 are required for apical F-actin and active myosin II localization and junction integrity. Furthermore, myosin II activity can function upstream of junction integrity during DLHP formation, and canonical Wnt signaling, an activator of zic gene transcription, is necessary for apical active myosin II localization, junction integrity and DLHP formation. We conclude that zic genes act downstream of Wnt signaling to control cytoskeletal organization, and possibly adhesion, during neurulation. This study identifies zic2a and zic5 as crucial players in the genetic network linking patterned gene expression to morphogenetic changes during neurulation, and strengthens the utility of the zebrafish midbrain as a NT morphogenesis model.

  8. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    PubMed Central

    Kim, Young Eun; Kim, Yu-na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-01-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein–protein interactions and tools to manipulate receptor clustering on live cell surfaces. PMID:25972078

  9. Visualizing Key Hinges and a Potential Major Source of Compliance in the Lever Arm of Myosin

    SciTech Connect

    J Brown; V Senthil Kumar; E ONeall-Hennessey; L Reshetnikova; H Robinson; M Nguyen-McCarty; A Szent-Gyorgyi; C Cohen

    2011-12-31

    We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

  10. Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

    SciTech Connect

    Brown, J.H.; Robinson, H.; Senthil Kumar, V. S.; O'Neall-Hennessey, E.; Reshetnikova, L.; Nguyen-McCarty, M.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-01-04

    We have determined the 2.3-{angstrom}-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch ('smooth') muscle. This structure reveals hinges that may function in the 'on' and 'off' states of myosin. The molecule adopts two different conformations about the heavy chain 'hook' and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 {angstrom}. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.

  11. From networks of protein interactions to networks of functional dependencies

    PubMed Central

    2012-01-01

    Background As protein-protein interactions connect proteins that participate in either the same or different functions, networks of interacting and functionally annotated proteins can be converted into process graphs of inter-dependent function nodes (each node corresponding to interacting proteins with the same functional annotation). However, as proteins have multiple annotations, the process graph is non-redundant, if only proteins participating directly in a given function are included in the related function node. Results Reasoning that topological features (e.g., clusters of highly inter-connected proteins) might help approaching structured and non-redundant understanding of molecular function, an algorithm was developed that prioritizes inclusion of proteins into the function nodes that best overlap protein clusters. Specifically, the algorithm identifies function nodes (and their mutual relations), based on the topological analysis of a protein interaction network, which can be related to various biological domains, such as cellular components (e.g., peroxisome and cellular bud) or biological processes (e.g., cell budding) of the model organism S. cerevisiae. Conclusions The method we have described allows converting a protein interaction network into a non-redundant process graph of inter-dependent function nodes. The examples we have described show that the resulting graph allows researchers to formulate testable hypotheses about dependencies among functions and the underlying mechanisms. PMID:22607727

  12. Consistent probabilistic outputs for protein function prediction

    PubMed Central

    Obozinski, Guillaume; Lanckriet, Gert; Grant, Charles; Jordan, Michael I; Noble, William Stafford

    2008-01-01

    In predicting hierarchical protein function annotations, such as terms in the Gene Ontology (GO), the simplest approach makes predictions for each term independently. However, this approach has the unfortunate consequence that the predictor may assign to a single protein a set of terms that are inconsistent with one another; for example, the predictor may assign a specific GO term to a given protein ('purine nucleotide binding') but not assign the parent term ('nucleotide binding'). Such predictions are difficult to interpret. In this work, we focus on methods for calibrating and combining independent predictions to obtain a set of probabilistic predictions that are consistent with the topology of the ontology. We call this procedure 'reconciliation'. We begin with a baseline method for predicting GO terms from a collection of data types using an ensemble of discriminative classifiers. We apply the method to a previously described benchmark data set, and we demonstrate that the resulting predictions are frequently inconsistent with the topology of the GO. We then consider 11 distinct reconciliation methods: three heuristic methods; four variants of a Bayesian network; an extension of logistic regression to the structured case; and three novel projection methods - isotonic regression and two variants of a Kullback-Leibler projection method. We evaluate each method in three different modes - per term, per protein and joint - corresponding to three types of prediction tasks. Although the principal goal of reconciliation is interpretability, it is important to assess whether interpretability comes at a cost in terms of precision and recall. Indeed, we find that many apparently reasonable reconciliation methods yield reconciled probabilities with significantly lower precision than the original, unreconciled estimates. On the other hand, we find that isotonic regression usually performs better than the underlying, unreconciled method, and almost never performs worse

  13. How special is the biochemical function of native proteins?

    PubMed

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2016-01-01

    Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation.

  14. How special is the biochemical function of native proteins?

    PubMed Central

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2016-01-01

    Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation. PMID:26962440

  15. Bayesian refinement of protein functional site matching

    PubMed Central

    Mardia, Kanti V; Nyirongo, Vysaul B; Green, Peter J; Gold, Nicola D; Westhead, David R

    2007-01-01

    Background Matching functional sites is a key problem for the understanding of protein function and evolution. The commonly used graph theoretic approach, and other related approaches, require adjustment of a matching distance threshold a priori according to the noise in atomic positions. This is difficult to pre-determine when matching sites related by varying evolutionary distances and crystallographic precision. Furthermore, sometimes the graph method is unable to identify alternative but important solutions in the neighbourhood of the distance based solution because of strict distance constraints. We consider the Bayesian approach to improve graph based solutions. In principle this approach applies to other methods with strict distance matching constraints. The Bayesian method can flexibly incorporate all types of prior information on specific binding sites (e.g. amino acid types) in contrast to combinatorial formulations. Results We present a new meta-algorithm for matching protein functional sites (active sites and ligand binding sites) based on an initial graph matching followed by refinement using a Markov chain Monte Carlo (MCMC) procedure. This procedure is an innovative extension to our recent work. The method accounts for the 3-dimensional structure of the site as well as the physico-chemical properties of the constituent amino acids. The MCMC procedure can lead to a significant increase in the number of significant matches compared to the graph method as measured independently by rigorously derived p-values. Conclusion MCMC refinement step is able to significantly improve graph based matches. We apply the method to matching NAD(P)(H) binding sites within single Rossmann fold families, between different families in the same superfamily, and in different folds. Within families sites are often well conserved, but there are examples where significant shape based matches do not retain similar amino acid chemistry, indicating that even within families the

  16. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  17. Protein-Polymer Functionalized Nanopatterned Surfaces

    NASA Astrophysics Data System (ADS)

    Wang, Haoyu; Akcora, Pinar

    2015-03-01

    Understanding and controlling the protein interactions with surfaces for biosensors and biomedical implants is a fundamental problem for biocompatible nanomaterial design. Proteins attached in ordered nanopores can exhibit superior biological activities compared to smooth microstructured surfaces. We developed heterogeneous and nanopatterned surfaces decorated with polymer brushes and proteins to control protein fates through elasticity. The heterogeneity of surfaces is controlled with well-defined chemistry, pattern size and geometry, stiffness of polymers and protein types. We will present our recent nanoindentation results on nanopatterned and biofunctionalized flat surfaces and discuss the pattern size effect on protein activity, hence conformation.

  18. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

    Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

  19. 14 CFR 25.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Structure Control Surface and System Loads... designed for inertia loads acting parallel to the hinge line. (b) In the absence of more rational data,...

  20. Drag-compensated, precision-powered hinge system

    NASA Technical Reports Server (NTRS)

    Jacquemin, G. G.; Rusk, S. J.

    1985-01-01

    The design of a high precision powered hinge is complicated by the unavoidable presence of parasitic drag torque resulting mainly from friction and transfer of power, signals, and fluids across the hinge. Regardless of the type of drive system selected, it is impossible to completely eliminate all parasitic drag. However, the mechanism described here comes very close to providing a drag free system. All sources of parasitic drag torque are collected on a shaft which is powered by an electric motor independent of the main hinge drive. Under control of a sensor, the electric motor applies a compensating torque equal to that of the parasitic drag torque, allowing the main hinge drive to operate in a practically drag free environment with very high positioning precision.

  1. 14 CFR 25.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... designed for inertia loads acting parallel to the hinge line. (b) In the absence of more rational data, the inertia loads may be assumed to be equal to KW, where— (1) K=24 for vertical surfaces; (2) K=12...

  2. 14 CFR 25.393 - Loads parallel to hinge line.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... designed for inertia loads acting parallel to the hinge line. (b) In the absence of more rational data, the inertia loads may be assumed to be equal to KW, where— (1) K=24 for vertical surfaces; (2) K=12...

  3. Lock 6 Detail of hinge stone with iron straps ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Lock 6 - Detail of hinge stone with iron straps and carved completion date (1830) located on ground at southeast corner of lock - Savannah & Ogeechee Barge Canal, Between Ogeechee & Savannah Rivers, Savannah, Chatham County, GA

  4. Principle of bio-inspired insect wing rotational hinge design

    NASA Astrophysics Data System (ADS)

    Fei, Fan

    A principle for designing and fabricating bio-inspired miniature artificial insect flapping wing using flexure rotational hinge design is presented. A systematic approach of selecting rotational hinge stiffness value is proposed. Based on the understanding of flapping wing aerodynamics, a dynamic simulation is constructed using the established quasi-steady model and the wing design. Simulations were performed to gain insight on how different parameters affect the wing rotational response. Based on system resonance a model to predict the optimal rotational hinge stiffness based on given wing parameter and flapping wing kinematic is proposed. By varying different wing parameters, the proposed method is shown to be applicable to a wide range of wing designs with different sizes and shapes. With the selected hinge stiffness value, aspects of the rotational joint design is discussed and an integrated wing-hinge structure design using laminated carbon fiber and polymer film is presented. Manufacturing process of such composite structure is developed to achieve high accuracy and repeatability. The yielded hinge stiffness is verified by measurements. To validate the proposed model, flapping wing experiments were conducted. A flapping actuation set up is built using DC motor and a controller is implemented on a microcontroller to track desired wing stroke kinematic. Wing stroke and rotation kinematic were extracted using a high speed camera and the lift generation is evaluated. A total of 49 flapping experiments were presented, experimental data shows good correlation with the model's prediction. With the wing rotational hinge stiffness designed so that the rotational resonant frequency is twice as the stroke frequency, the resulting wing rotation generates near optimal lift. With further simulation, the proposed model shows low sensitivity to wing parameter variation. As a result, giving a design parameter of a flapping wing robot platform, the proposed principle can

  5. Evolutionary and functional diversity of coronin proteins.

    PubMed

    Xavier, Charles-Peter; Eichinger, Ludwig; Fernandez, M Pilar; Morgan, Reginald O; Clemen, Christoph S

    2008-01-01

    This chapter discusses various aspects of coronin phylogeny, structure and function that are of specific interest. Two subfamilies of ancient coronins of unicellular pathogens such as Entamoeba, Trypanosoma, Leishmania and Acanthamoeba as well as of Plasmodium, Babesia, and Trichomonas are presented in the first two sections. Their coronins generally bind to F-actin and apparently are involved in proliferation, locomotion and phagocytosis. However, there are so far no studies addressing a putative role of coronin in the virulence of these pathogens. The following section delineates genetic anomalies like the chimeric coronin-fusion products with pelckstrin homology and gelsolin domains that are found in amoeba. Moreover, most nonvertebrate metazoa appear to encode CRN8, CRN9 and CRN7 representatives (for these coronin symbols see Chapter 2), but in e.g., Drosophila melanogaster and Caenorhabditis elegans a CRN9 is missing. The forth section deals with the evolutionary expansion of vertebrate coronins. Experimental data on the F-actin binding CRN2 of Xenopus (Xcoronin) including a Cdc42/Rac interactive binding (CRIB) motif that is also present in other members of the coronin protein family are discussed. Xenopus laevis represents a case for the expansion of the seven vertebrate coronins due to tetraploidization events. Other examples for a change in the number of coronin paralogs are zebrafish and birds, but (coronin) gene duplication events also occurred in unicellular protozoa. The fifth section of this chapter briefly summarizes three different cellular processes in which CRN4/CORO1A is involved, namely actin-binding, superoxide generation and Ca(2+)-signaling and refers to the largely unexplored mammalian coronins CRN5/CORO2A and CRN6/CORO2B, the latter binding to vinculin. The final section discusses how, by unveiling the aspects of coronin function in organisms reported so far, one can trace a remarkable evolution and diversity in their individual roles

  6. Computational approaches for rational design of proteins with novel functionalities.

    PubMed

    Tiwari, Manish Kumar; Singh, Ranjitha; Singh, Raushan Kumar; Kim, In-Won; Lee, Jung-Kul

    2012-01-01

    Proteins are the most multifaceted macromolecules in living systems and have various important functions, including structural, catalytic, sensory, and regulatory functions. Rational design of enzymes is a great challenge to our understanding of protein structure and physical chemistry and has numerous potential applications. Protein design algorithms have been applied to design or engineer proteins that fold, fold faster, catalyze, catalyze faster, signal, and adopt preferred conformational states. The field of de novo protein design, although only a few decades old, is beginning to produce exciting results. Developments in this field are already having a significant impact on biotechnology and chemical biology. The application of powerful computational methods for functional protein designing has recently succeeded at engineering target activities. Here, we review recently reported de novo functional proteins that were developed using various protein design approaches, including rational design, computational optimization, and selection from combinatorial libraries, highlighting recent advances and successes.

  7. Design and analysis of a high-accuracy flexure hinge.

    PubMed

    Liu, Min; Zhang, Xianmin; Fatikow, Sergej

    2016-05-01

    This paper designs and analyzes a new kind of flexure hinge obtained by using a topology optimization approach, namely, a quasi-V-shaped flexure hinge (QVFH). Flexure hinges are formed by three segments: the left and right segments with convex shapes and the middle segment with straight line. According to the results of topology optimization, the curve equations of profiles of the flexure hinges are developed by numerical fitting. The in-plane dimensionless compliance equations of the flexure hinges are derived based on Castigliano's second theorem. The accuracy of rotation, which is denoted by the compliance of the center of rotation that deviates from the midpoint, is derived. The equations for evaluating the maximum stresses are also provided. These dimensionless equations are verified by finite element analysis and experimentation. The analytical results are within 8% uncertainty compared to the finite element analysis results and within 9% uncertainty compared to the experimental measurement data. Compared with the filleted V-shaped flexure hinge, the QVFH has a higher accuracy of rotation and better ability of preserving the center of rotation position but smaller compliance. PMID:27250469

  8. Applications in high-content functional protein microarrays.

    PubMed

    Moore, Cedric D; Ajala, Olutobi Z; Zhu, Heng

    2016-02-01

    Protein microarray technology provides a versatile platform for characterization of hundreds to thousands of proteins in a parallel and high-throughput manner. Over the last decade, applications of functional protein microarrays in particular have flourished in studying protein function at a systems level and have led to the construction of networks and pathways describing these functions. Relevant areas of research include the detection of various binding properties of proteins, the study of enzyme-substrate relationships, the analysis of host-microbe interactions, and profiling antibody specificity. In addition, discovery of novel biomarkers in autoimmune diseases and cancers is emerging as a major clinical application of functional protein microarrays. In this review, we will summarize the recent advances of functional protein microarrays in both basic and clinical applications. PMID:26599287

  9. Functional innovation from changes in protein domains and their combinations.

    PubMed

    Lees, Jonathan G; Dawson, Natalie L; Sillitoe, Ian; Orengo, Christine A

    2016-06-01

    Domains are the functional building blocks of proteins. In this work we discuss how domains can contribute to the evolution of new functions. Domains themselves can evolve through various mechanisms, altering their intrinsic function. Domains can also facilitate functional innovations by combining with other domains to make novel proteins. We discuss the mechanisms by which domain and domain combinations support functional innovations. We highlight interesting examples where changes in domain combination promote changes at the domain level. PMID:27309309

  10. Functional protein aggregates: just the tip of the iceberg.

    PubMed

    Villaverde, Antonio; Corchero, José Luis; Seras-Franzoso, Joaquin; Garcia-Fruitós, Elena

    2015-01-01

    An increasing number of both prokaryotic and eukaryotic cell types are being adapted as platforms for recombinant protein production. The overproduction of proteins in such expression systems leads to the formation of insoluble protein-based aggregates. Although these protein clusters have been poorly studied in most of the eukaryotic systems, aggregates formed in E. coli, named inclusion bodies (IBs), have been deeply characterized in the last decades. Contrary to the general belief, an important fraction of the protein embedded in IB is functional, showing promise in biocatalysis, regenerative medicine and cell therapy. Thus, the exploration of all these functional protein clusters would largely expand their potential in both pharma and biotech industry.

  11. Ion-dipole interactions and their functions in proteins.

    PubMed

    Sippel, Katherine H; Quiocho, Florante A

    2015-07-01

    Ion-dipole interactions in biological macromolecules are formed between atomic or molecular ions and neutral protein dipolar groups through either hydrogen bond or coordination. Since their discovery 30 years ago, these interactions have proven to be a frequent occurrence in protein structures, appearing in everything from transporters and ion channels to enzyme active sites to protein-protein interfaces. However, their significance and roles in protein functions are largely underappreciated. We performed PDB data mining to identify a sampling of proteins that possess these interactions. In this review, we will define the ion-dipole interaction and discuss several prominent examples of their functional roles in nature.

  12. Exploring the evolution of protein function in Archaea

    PubMed Central

    2012-01-01

    Background Despite recent progress in studies of the evolution of protein function, the questions what were the first functional protein domains and what were their basic building blocks remain unresolved. Previously, we introduced the concept of elementary functional loops (EFLs), which are the functional units of enzymes that provide elementary reactions in biochemical transformations. They are presumably descendants of primordial catalytic peptides. Results We analyzed distant evolutionary connections between protein functions in Archaea based on the EFLs comprising them. We show examples of the involvement of EFLs in new functional domains, as well as reutilization of EFLs and functional domains in building multidomain structures and protein complexes. Conclusions Our analysis of the archaeal superkingdom yields the dominating mechanisms in different periods of protein evolution, which resulted in several levels of the organization of biochemical function. First, functional domains emerged as combinations of prebiotic peptides with the very basic functions, such as nucleotide/phosphate and metal cofactor binding. Second, domain recombination brought to the evolutionary scene the multidomain proteins and complexes. Later, reutilization and de novo design of functional domains and elementary functional loops complemented evolution of protein function. PMID:22646318

  13. Sortase A-mediated multi-functionalization of protein nanoparticles.

    PubMed

    Chen, Qi; Sun, Qing; Molino, Nicholas M; Wang, Szu-Wen; Boder, Eric T; Chen, Wilfred

    2015-08-01

    We report here a new strategy to enable fast, covalent, and site-directed functionalization of protein nanoparticles using Sortase A-mediated ligation using functional proteins ranging from monomeric to large tetrameric structures. Easy purification of the modified E2 nanoparticles is achieved by functionalization with a thermo-responsive elastin-like-peptide. The resulting protein nanoparticles remained intact and active even after repeated phase transitions, suggesting their use in biocatalysis, biosensing, and imaging applications.

  14. Functional properties of select edible oilseed proteins.

    PubMed

    Sharma, Girdhari M; Su, Mengna; Joshi, Aditya U; Roux, Kenneth H; Sathe, Shridhar K

    2010-05-12

    Borate saline buffer (0.1 M, pH 8.45) solubilized proteins from almond, Brazil nut, cashew nut, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean seeds were prepared from the corresponding defatted flour. The yield was in the range from 10.6% (macadamia) to 27.4% (almond). The protein content, on a dry weight basis, of the lyophilized preparations ranged from 69.23% (pine nut) to 94.80% (soybean). Isolated proteins from Brazil nut had the lightest and hazelnut the darkest color. Isolated proteins exhibited good solubility in aqueous media. Foaming capacity (<40% overrun) and stability (<1 h) of the isolated proteins were poor to fair. Almond proteins had the highest viscosity among the tested proteins. Oil-holding capacity of the isolated proteins ranged from 2.8 (macadamia) to 7 (soybean) g of oil/g of protein. Least gelation concentrations (% w/v) for almond, Brazil nut, cashew, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean were, respectively, 6, 8, 8, 12, 20, 12, 10, 14, 14, and 16.

  15. Functional properties of select edible oilseed proteins.

    PubMed

    Sharma, Girdhari M; Su, Mengna; Joshi, Aditya U; Roux, Kenneth H; Sathe, Shridhar K

    2010-05-12

    Borate saline buffer (0.1 M, pH 8.45) solubilized proteins from almond, Brazil nut, cashew nut, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean seeds were prepared from the corresponding defatted flour. The yield was in the range from 10.6% (macadamia) to 27.4% (almond). The protein content, on a dry weight basis, of the lyophilized preparations ranged from 69.23% (pine nut) to 94.80% (soybean). Isolated proteins from Brazil nut had the lightest and hazelnut the darkest color. Isolated proteins exhibited good solubility in aqueous media. Foaming capacity (<40% overrun) and stability (<1 h) of the isolated proteins were poor to fair. Almond proteins had the highest viscosity among the tested proteins. Oil-holding capacity of the isolated proteins ranged from 2.8 (macadamia) to 7 (soybean) g of oil/g of protein. Least gelation concentrations (% w/v) for almond, Brazil nut, cashew, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean were, respectively, 6, 8, 8, 12, 20, 12, 10, 14, 14, and 16. PMID:20201552

  16. Under-folded proteins: Conformational ensembles and their roles in protein folding, function, and pathogenesis.

    PubMed

    Uversky, Vladimir N

    2013-11-01

    For decades, protein function was intimately linked to the presence of a unique, aperiodic crystal-like structure in a functional protein. The two only places for conformational ensembles of under-folded (or partially folded) protein forms in this picture were either the end points of the protein denaturation processes or transiently populated folding intermediates. Recent years witnessed dramatic change in this perception and conformational ensembles, which the under-folded proteins are, have moved from the shadow. Accumulated to date data suggest that a protein can exist in at least three global forms-functional and folded, functional and intrinsically disordered (nonfolded), and nonfunctional and misfolded/aggregated. Under-folded protein states are crucial for each of these forms, serving as important folding intermediates of ordered proteins, or as functional states of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), or as pathology triggers of misfolded proteins. Based on these observations, conformational ensembles of under-folded proteins can be classified as transient (folding and misfolding intermediates) and permanent (IDPs and stable misfolded proteins). Permanently under-folded proteins can further be split into intentionally designed (IDPs and IDPRs) and unintentionally designed (misfolded proteins). Although intrinsic flexibility, dynamics, and pliability are crucial for all under-folded proteins, the different categories of under-foldedness are differently encoded in protein amino acid sequences.

  17. The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study

    PubMed Central

    Sun, Lili; Tabaka, Marcin; Hou, Sen; Li, Lin; Burdzy, Krzysztof; Aksimentiev, Aleksei; Maffeo, Christopher; Zhang, Xuzhu; Holyst, Robert

    2016-01-01

    LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex. PMID:27008630

  18. Printing Proteins as Microarrays for High-Throughput Function Determination

    NASA Astrophysics Data System (ADS)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  19. Nonlinear Models for Protein Folding and Function

    NASA Astrophysics Data System (ADS)

    Cruzeiro, L.

    Earlier a specific kinetic process for reproducible protein folding was proposed according to which the nascent chain is helical and the first step in in vivo protein folding is the bending of the initial helix at specific amino acid sites. Here the theoretical feasibility of this kinetic process is tested. To that end, two proteins, one belonging to the mainly α class and the other belonging to the α/β class, are selected and targeted molecular dynamics is applied to generate folding pathways for those two proteins, starting from two well defined initial conformations: a fully extended and a α-helical conformation. Not only are the native states closer to an initial helical structure for both proteins but also the pathways from the α-helical initial conformation to the native state have lower potential energy than the pathways that start from the fully extended conformation. For the α/β protein, 30% (40%) of the pathways from an initial α-helix (fully extended) structure lead to unentangled native folds, a success rate that can be increased to 85% by the introduction of a putative intermediate structure. These results lend support to the kinetic process proposed and open up a new direction in which to look for a solution to the protein folding problem. The chapter ends with a section that emphasizes the formal similarities between the dynamics quantum vibrational excited states in proteins and electrons in nonlinear lattices.

  20. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  1. Recent approaches in physical modification of protein functionality.

    PubMed

    Mirmoghtadaie, Leila; Shojaee Aliabadi, Saeedeh; Hosseini, Seyede Marzieh

    2016-05-15

    Today, there is a growing demand for novel technologies, such as high hydrostatic pressure, irradiation, ultrasound, filtration, supercritical carbon dioxide, plasma technology, and electrical methods, which are not based on chemicals or heat treatment for modifying ingredient functionality and extending product shelf life. Proteins are essential components in many food processes, and provide various functions in food quality and stability. They can create interfacial films that stabilize emulsions and foams as well as interact to make networks that play key roles in gel and edible film production. These properties of protein are referred to as 'protein functionality', because they can be modified by different processing. The common protein modification (chemical, enzymatic and physical) methods have strong effects on the structure and functionality of food proteins. Furthermore, novel technologies can modify protein structure and functional properties that will be reviewed in this study.

  2. Recent approaches in physical modification of protein functionality.

    PubMed

    Mirmoghtadaie, Leila; Shojaee Aliabadi, Saeedeh; Hosseini, Seyede Marzieh

    2016-05-15

    Today, there is a growing demand for novel technologies, such as high hydrostatic pressure, irradiation, ultrasound, filtration, supercritical carbon dioxide, plasma technology, and electrical methods, which are not based on chemicals or heat treatment for modifying ingredient functionality and extending product shelf life. Proteins are essential components in many food processes, and provide various functions in food quality and stability. They can create interfacial films that stabilize emulsions and foams as well as interact to make networks that play key roles in gel and edible film production. These properties of protein are referred to as 'protein functionality', because they can be modified by different processing. The common protein modification (chemical, enzymatic and physical) methods have strong effects on the structure and functionality of food proteins. Furthermore, novel technologies can modify protein structure and functional properties that will be reviewed in this study. PMID:26776016

  3. Computer-Based Screening of Functional Conformers of Proteins

    PubMed Central

    Montiel Molina, Héctor Marlosti; Millán-Pacheco, César; Pastor, Nina; del Rio, Gabriel

    2008-01-01

    A long-standing goal in biology is to establish the link between function, structure, and dynamics of proteins. Considering that protein function at the molecular level is understood by the ability of proteins to bind to other molecules, the limited structural data of proteins in association with other bio-molecules represents a major hurdle to understanding protein function at the structural level. Recent reports show that protein function can be linked to protein structure and dynamics through network centrality analysis, suggesting that the structures of proteins bound to natural ligands may be inferred computationally. In the present work, a new method is described to discriminate protein conformations relevant to the specific recognition of a ligand. The method relies on a scoring system that matches critical residues with central residues in different structures of a given protein. Central residues are the most traversed residues with the same frequency in networks derived from protein structures. We tested our method in a set of 24 different proteins and more than 260,000 structures of these in the absence of a ligand or bound to it. To illustrate the usefulness of our method in the study of the structure/dynamics/function relationship of proteins, we analyzed mutants of the yeast TATA-binding protein with impaired DNA binding. Our results indicate that critical residues for an interaction are preferentially found as central residues of protein structures in complex with a ligand. Thus, our scoring system effectively distinguishes protein conformations relevant to the function of interest. PMID:18463705

  4. Method for printing functional protein microarrays

    NASA Technical Reports Server (NTRS)

    Delehanty, James B.; Ligler, Frances S.

    2003-01-01

    Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.

  5. The auto-inhibitory role of the EPAC hinge helix as mapped by NMR.

    PubMed

    Selvaratnam, Rajeevan; Mazhab-Jafari, Mohammad T; Das, Rahul; Melacini, Giuseppe

    2012-01-01

    The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.

  6. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  7. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators. PMID:27515397

  8. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.

  9. Structure and function of antifreeze proteins.

    PubMed Central

    Davies, Peter L; Baardsnes, Jason; Kuiper, Michael J; Walker, Virginia K

    2002-01-01

    High-resolution three-dimensional structures are now available for four of seven non-homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been defined by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic -- more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts. PMID:12171656

  10. Ion–dipole interactions and their functions in proteins

    PubMed Central

    Sippel, Katherine H; Quiocho, Florante A

    2015-01-01

    Ion–dipole interactions in biological macromolecules are formed between atomic or molecular ions and neutral protein dipolar groups through either hydrogen bond or coordination. Since their discovery 30 years ago, these interactions have proven to be a frequent occurrence in protein structures, appearing in everything from transporters and ion channels to enzyme active sites to protein–protein interfaces. However, their significance and roles in protein functions are largely underappreciated. We performed PDB data mining to identify a sampling of proteins that possess these interactions. In this review, we will define the ion–dipole interaction and discuss several prominent examples of their functional roles in nature. PMID:25866296

  11. Internal symmetry in protein structures: prevalence, functional relevance and evolution.

    PubMed

    Balaji, Santhanam

    2015-06-01

    Symmetry has been found at various levels of biological organization in the protein structural universe. Numerous evolutionary studies have proposed connections between internal symmetry within protein tertiary structures, quaternary associations and protein functions. Recent computational methods, such as SymD and CE-Symm, facilitate a large-scale detection of internal symmetry in protein structures. Based on the results from these methods, about 20% of SCOP folds, superfamilies and families are estimated to have structures with internal symmetry (Figure 1d). All-β and membrane proteins fold classes contain a relatively high number of unique instances of internal symmetry. In addition to the axis of symmetry, anecdotal evidence suggests that, the region of connection or contact between symmetric units could coincide with functionally relevant sites within a fold. General principles that underlie protein internal symmetry and their connections to protein structural integrity and functions remain to be elucidated.

  12. Computational design of proteins with novel structure and functions

    NASA Astrophysics Data System (ADS)

    Wei, Yang; Lu-Hua, Lai

    2016-01-01

    Computational design of proteins is a relatively new field, where scientists search the enormous sequence space for sequences that can fold into desired structure and perform desired functions. With the computational approach, proteins can be designed, for example, as regulators of biological processes, novel enzymes, or as biotherapeutics. These approaches not only provide valuable information for understanding of sequence-structure-function relations in proteins, but also hold promise for applications to protein engineering and biomedical research. In this review, we briefly introduce the rationale for computational protein design, then summarize the recent progress in this field, including de novo protein design, enzyme design, and design of protein-protein interactions. Challenges and future prospects of this field are also discussed. Project supported by the National Basic Research Program of China (Grant No. 2015CB910300), the National High Technology Research and Development Program of China (Grant No. 2012AA020308), and the National Natural Science Foundation of China (Grant No. 11021463).

  13. Post-translational control of protein function with light using a LOV-intein fusion protein.

    PubMed

    Jones, D C; Mistry, I N; Tavassoli, A

    2016-04-01

    Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells. PMID:26940144

  14. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  15. Protein Carbonylation and Adipocyte Mitochondrial Function*

    PubMed Central

    Curtis, Jessica M.; Hahn, Wendy S.; Stone, Matthew D.; Inda, Jacob J.; Droullard, David J.; Kuzmicic, Jovan P.; Donoghue, Margaret A.; Long, Eric K.; Armien, Anibal G.; Lavandero, Sergio; Arriaga, Edgar; Griffin, Timothy J.; Bernlohr, David A.

    2012-01-01

    Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte. PMID:22822087

  16. A Survey of Computational Intelligence Techniques in Protein Function Prediction

    PubMed Central

    Tiwari, Arvind Kumar; Srivastava, Rajeev

    2014-01-01

    During the past, there was a massive growth of knowledge of unknown proteins with the advancement of high throughput microarray technologies. Protein function prediction is the most challenging problem in bioinformatics. In the past, the homology based approaches were used to predict the protein function, but they failed when a new protein was different from the previous one. Therefore, to alleviate the problems associated with homology based traditional approaches, numerous computational intelligence techniques have been proposed in the recent past. This paper presents a state-of-the-art comprehensive review of various computational intelligence techniques for protein function predictions using sequence, structure, protein-protein interaction network, and gene expression data used in wide areas of applications such as prediction of DNA and RNA binding sites, subcellular localization, enzyme functions, signal peptides, catalytic residues, nuclear/G-protein coupled receptors, membrane proteins, and pathway analysis from gene expression datasets. This paper also summarizes the result obtained by many researchers to solve these problems by using computational intelligence techniques with appropriate datasets to improve the prediction performance. The summary shows that ensemble classifiers and integration of multiple heterogeneous data are useful for protein function prediction. PMID:25574395

  17. Bioinformatics pipeline for functional identification and characterization of proteins

    NASA Astrophysics Data System (ADS)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; Krzywkowski, Tomasz; Świerkula, Katarzyna; PlÄ der, Wojciech; Przybecki, Zbigniew

    2015-09-01

    The new sequencing methods, called Next Generation Sequencing gives an opportunity to possess a vast amount of data in short time. This data requires structural and functional annotation. Functional identification and characterization of predicted proteins could be done by in silico approches, thanks to a numerous computational tools available nowadays. However, there is a need to confirm the results of proteins function prediction using different programs and comparing the results or confirm experimentally. Here we present a bioinformatics pipeline for structural and functional annotation of proteins.

  18. Composition and functional properties of Lupinus campestris protein isolates.

    PubMed

    Rodríguez-Ambriz, S L; Martínez-Ayala, A L; Millán, F; Dávila-Ortíz, G

    2005-09-01

    Protein isolates from L. campestris and soybean seeds were prepared using isoelectric precipitation (PI) and micellization (MI) procedures. The amount of protein recovered was considerably higher with the isoelectric precipitation than with the micellization procedure (60% and 30%, respectively). Protein contents were higher than 90% in protein isolates. Antinutritional factors content (alkaloids, lectins, and tannins) were reduced to innocuous levels after protein isolate preparation. Minimum protein solubility for the precipitated lupin protein isolate (LPI) was at pH 4.0, and between pH 4 and 6 for the micellized lupin protein isolate (LMI), increasing at both extremes of the pH scale. Water absorption for the LMI was 1.3 ml/g of protein and its oil absorption 2.2 ml/g of protein. The LPI had 1.7 ml/g of protein in both water and oil absorption. Foaming capacity and stability was pH-dependent. Foaming capacity was higher at pH 2 and lower near the protein isoelectric points. Minimum protein concentration for gelation in LMI was 8% w/v at pH 4, while for LPI was 6% at pH 4 and 6. Amino acid composition in L. campestris flour and protein isolates was high in lysine and low in methionine. Most of the essential amino acids in lupin protein isolates were at acceptable levels compared to a reference pattern for infants and adults. The electrophoretic pattern of both protein isolates showed three bands with different mobilities, suggesting that the protein fractions belong to alpha-conglutin (11S-like protein), beta-conglutin (7S-like protein) and gamma-conglutin. It is proven that some of the functional properties of L. campestris protein isolates are similar to those soybean protein isolates recovered under equal conditions. PMID:16187011

  19. Adaptive fiber optics collimator based on flexible hinges.

    PubMed

    Zhi, Dong; Ma, Yanxing; Ma, Pengfei; Si, Lei; Wang, Xiaolin; Zhou, Pu

    2014-08-20

    In this manuscript, we present a new design for an adaptive fiber optics collimator (AFOC) based on flexible hinges by using piezoelectric stacks actuators for X-Y displacement. Different from traditional AFOC, the new structure is based on flexible hinges to drive the fiber end cap instead of naked fiber. We fabricated a real AFOC based on flexible hinges, and the end cap's deviation and resonance frequency of the device were measured. Experimental results show that this new AFOC can provide fast control of tip-tilt deviation of the laser beam emitting from the end cap. As a result, the fiber end cap can support much higher power than naked fiber, which makes the new structure ideal for tip-tilt controlling in a high-power fiber laser system.

  20. Gas turbine combustor exit piece with hinged connections

    DOEpatents

    Charron, Richard C.; Pankey, William W.

    2016-04-26

    An exit piece (66) with an inlet throat (67) that conducts a combustion gas flow (36A) in a path (82) from a combustor (63) to an annular chamber (68) that feeds the first blade section (37) of a gas turbine (26). The exit piece further includes an outlet portion (69) that forms a circumferential segment of the annular chamber. The outlet portion interconnects with adjacent outlet portions by hinges (78A, 78B, 80A, 80B). Each hinge may have a hinge axis (82A, 82B) parallel to a centerline (21) of the turbine. Respective gas flows (36A) are configured by an assembly (60) of the exit pieces to converge on the feed chamber (68) into a uniform helical flow that drives the first blade section with minimal circumferential variations in force.

  1. Intracellular delivery of functional proteins via decoration with transporter peptides.

    PubMed

    Siprashvili, Zurab; Reuter, Jason A; Khavari, Paul A

    2004-05-01

    Despite numerous attractive intracellular targets, protein therapeutics have been principally confined to the extracellular space due to the lack of a straightforward way to deliver functional polypeptides to the cell interior. Peptide sequences facilitating intracellular protein delivery have been identified; however, current strategies to apply them require problematic steps, such as generation of new in-frame fusion proteins, covalent chemical conjugation, and denaturation. We have developed a new approach to protein transfer into cells and tissues that relies on single-step decoration by cysteine-flanked, arginine-rich transporter peptides. This approach facilitated cell and tissue delivery of a variety of functional proteins, including antibodies and enzymes. Decoration with transporter peptides thus provides an attractive general means of intracellular delivery of functional proteins in vitro and in tissue.

  2. Infrared Structural Biology: Detect Functionally Important Structural Motions of Proteins

    NASA Astrophysics Data System (ADS)

    Xie, Aihua

    Proteins are dynamic. Lack of dynamic structures of proteins hampers our understanding of protein functions. Infrared structural biology (IRSB) is an emerging technology. There are several advantages of IRSB for mechanistic studies of proteins: (1) its excellent dynamic range (detecting structural motions from picoseconds to >= seconds); (2) its high structural sensitivity (detect tiny but functionally important structural motions such as proton transfer and changes in hydrogen bonding interaction); (3) its ability to detect different structural motions simultaneously. Successful development of infrared structural biology demands not only new experimental techniques (from infrared technologies to chemical synthesis and cell biology), but also new data processing (how to translate infrared signals into quantitative structural information of proteins). These topics will be discussed as well as examples of how to use IRSB to study structure-function relationship of proteins. This work was supported by NSF DBI1338097 and OCAST HR10-078.

  3. Analysis of Arf GTP-binding Protein Function in Cells

    PubMed Central

    Cohen, Lee Ann; Donaldson, Julie G.

    2010-01-01

    This unit describes techniques and approaches that can be used to study the functions of the ADP-ribosylation factor (Arf) GTP-binding proteins in cells. There are 6 mammalian Arfs and many more Arf-like proteins (Arls) and these proteins are conserved in eukaryotes from yeast to man. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze GTP binding and hydrolysis respectively. Here we describe approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells (Protocol 1). We also provide a simple protocol for measuring activation (GTP-binding) of specific Arf proteins in cells using a pull-down assay (Protocol 2). We then discuss approaches that can be taken to assess function of GEFs and GAPs in cells (Protocol 3). PMID:20853342

  4. Canola proteins for human consumption: extraction, profile, and functional properties.

    PubMed

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies.

  5. Protein function prediction using guilty by association from interaction networks.

    PubMed

    Piovesan, Damiano; Giollo, Manuel; Ferrari, Carlo; Tosatto, Silvio C E

    2015-12-01

    Protein function prediction from sequence using the Gene Ontology (GO) classification is useful in many biological problems. It has recently attracted increasing interest, thanks in part to the Critical Assessment of Function Annotation (CAFA) challenge. In this paper, we introduce Guilty by Association on STRING (GAS), a tool to predict protein function exploiting protein-protein interaction networks without sequence similarity. The assumption is that whenever a protein interacts with other proteins, it is part of the same biological process and located in the same cellular compartment. GAS retrieves interaction partners of a query protein from the STRING database and measures enrichment of the associated functional annotations to generate a sorted list of putative functions. A performance evaluation based on CAFA metrics and a fair comparison with optimized BLAST similarity searches is provided. The consensus of GAS and BLAST is shown to improve overall performance. The PPI approach is shown to outperform similarity searches for biological process and cellular compartment GO predictions. Moreover, an analysis of the best practices to exploit protein-protein interaction networks is also provided.

  6. Canola Proteins for Human Consumption: Extraction, Profile, and Functional Properties

    PubMed Central

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies. PMID:21535703

  7. The Structure and Function of Non-Collagenous Bone Proteins

    NASA Technical Reports Server (NTRS)

    Hook, Magnus; McQuillan, David J.

    1997-01-01

    The research done under the cooperative research agreement for the project titled 'The structure and function of non-collagenous bone proteins' represented the first phase of an ongoing program to define the structural and functional relationships of the principal noncollagenous proteins in bone. An ultimate goal of this research is to enable design and execution of useful pharmacological compounds that will have a beneficial effect in treatment of osteoporosis, both land-based and induced by long-duration space travel. The goals of the now complete first phase were as follows: 1. Establish and/or develop powerful recombinant protein expression systems; 2. Develop and refine isolation and purification of recombinant proteins; 3. Express wild-type non-collagenous bone proteins; 4. Express site-specific mutant proteins and domains of wild-type proteins to enhance likelihood of crystal formation for subsequent solution of structure.

  8. Improvement in protein functional site prediction by distinguishing structural and functional constraints on protein family evolution using computational design.

    PubMed

    Cheng, Gong; Qian, Bin; Samudrala, Ram; Baker, David

    2005-01-01

    The prediction of functional sites in newly solved protein structures is a challenge for computational structural biology. Most methods for approaching this problem use evolutionary conservation as the primary indicator of the location of functional sites. However, sequence conservation reflects not only evolutionary selection at functional sites to maintain protein function, but also selection throughout the protein to maintain the stability of the folded state. To disentangle sequence conservation due to protein functional constraints from sequence conservation due to protein structural constraints, we use all atom computational protein design methodology to predict sequence profiles expected under solely structural constraints, and to compute the free energy difference between the naturally occurring amino acid and the lowest free energy amino acid at each position. We show that functional sites are more likely than non-functional sites to have computed sequence profiles which differ significantly from the naturally occurring sequence profiles and to have residues with sub-optimal free energies, and that incorporation of these two measures improves sequence based prediction of protein functional sites. The combined sequence and structure based functional site prediction method has been implemented in a publicly available web server.

  9. Protein Structure and Function Prediction Using I-TASSER.

    PubMed

    Yang, Jianyi; Zhang, Yang

    2015-01-01

    I-TASSER is a hierarchical protocol for automated protein structure prediction and structure-based function annotation. Starting from the amino acid sequence of target proteins, I-TASSER first generates full-length atomic structural models from multiple threading alignments and iterative structural assembly simulations followed by atomic-level structure refinement. The biological functions of the protein, including ligand-binding sites, enzyme commission number, and gene ontology terms, are then inferred from known protein function databases based on sequence and structure profile comparisons. I-TASSER is freely available as both an on-line server and a stand-alone package. This unit describes how to use the I-TASSER protocol to generate structure and function prediction and how to interpret the prediction results, as well as alternative approaches for further improving the I-TASSER modeling quality for distant-homologous and multi-domain protein targets.

  10. Collective Dynamics Differentiates Functional Divergence in Protein Evolution

    PubMed Central

    Glembo, Tyler J.; Farrell, Daniel W.; Gerek, Z. Nevin; Thorpe, M. F.; Ozkan, S. Banu

    2012-01-01

    Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function. PMID:22479170

  11. Extra-nuclear starbursts: young luminous Hinge clumps in interacting galaxies

    SciTech Connect

    Smith, Beverly J.; Giroux, Mark L.; Soria, Roberto; Struck, Curtis; Swartz, Douglas A.; Yukita, Mihoko E-mail: girouxm@etsu.edu E-mail: curt@iastate.edu

    2014-03-01

    Hinge clumps are luminous knots of star formation near the base of tidal features in some interacting galaxies. We use archival Hubble Space Telescope (HST) UV/optical/IR images and Chandra X-ray maps along with Galaxy Evolution Explorer UV, Spitzer IR, and ground-based optical/near-IR images to investigate the star forming properties in a sample of 12 hinge clumps in five interacting galaxies. The most extreme of these hinge clumps have star formation rates of 1-9 M {sub ☉} yr{sup –1}, comparable to or larger than the 'overlap' region of intense star formation between the two disks of the colliding galaxy system the Antennae. In the HST images, we have found remarkably large and luminous sources at the centers of these hinge clumps. These objects are much larger and more luminous than typical 'super star clusters' in interacting galaxies, and are sometimes embedded in a linear ridge of fainter star clusters, consistent with star formation along a narrow caustic. These central sources have FWHM diameters of ∼70 pc, compared to ∼3 pc in 'ordinary' super star clusters. Their absolute I magnitudes range from M{sub I} ∼ – 12.2 to –16.5; thus, if they are individual star clusters they would lie near the top of the 'super star cluster' luminosity function of star clusters. These sources may not be individual star clusters, but instead may be tightly packed groups of clusters that are blended together in the HST images. Comparison to population synthesis modeling indicates that the hinge clumps contain a range of stellar ages. This is consistent with expectations based on models of galaxy interactions, which suggest that star formation may be prolonged in these regions. In the Chandra images, we have found strong X-ray emission from several of these hinge clumps. In most cases, this emission is well-resolved with Chandra and has a thermal X-ray spectrum, thus it is likely due to hot gas associated with the star formation. The ratio of the extinction

  12. Mapping the Function of Polycomb Proteins.

    PubMed

    Pasini, Diego

    2016-01-01

    Polycomb group (PcG) proteins are master regulators of proliferation and development that play essential roles in human pathologies including cancers. PcGs act as gatekeepers of cellular identity, maintaining repression of a multitude of target genes. However, these properties have only been recently uncovered thanks to technological advances, first of all chromatin immunoprecipitations (ChIP), that allowed a systematic characterization of the activity of these factors in an unbiased manner at a genome-wide level. Using PcG protein as example, this chapter introduces the readers to the use of chromatin analysis (ChIP assays and replication timing) and how to move these approaches to a level of genome-wide interpretation. PMID:27659970

  13. Protein mislocalization: mechanisms, functions and clinical applications in cancer

    PubMed Central

    Wang, Xiaohong; Li, Shulin

    2014-01-01

    The changes from normal cells to cancer cells are primarily regulated by genome instability, which foster hallmark functions of cancer through multiple mechanisms including protein mislocalization. Mislocalization of these proteins, including oncoproteins, tumor suppressors, and other cancer-related proteins, can interfere with normal cellular function and cooperatively drive tumor development and metastasis. This review describes the cancer-related effects of protein subcellular mislocalization, the related mislocalization mechanisms, and the potential application of this knowledge to cancer diagnosis, prognosis, and therapy. PMID:24709009

  14. PIRSF family classification system for protein functional and evolutionary analysis.

    PubMed

    Nikolskaya, Anastasia N; Arighi, Cecilia N; Huang, Hongzhan; Barker, Winona C; Wu, Cathy H

    2006-01-01

    The PIRSF protein classification system (http://pir.georgetown.edu/pirsf/) reflects evolutionary relationships of full-length proteins and domains. The primary PIRSF classification unit is the homeomorphic family, whose members are both homologous (evolved from a common ancestor) and homeomorphic (sharing full-length sequence similarity and a common domain architecture). PIRSF families are curated systematically based on literature review and integrative sequence and functional analysis, including sequence and structure similarity, domain architecture, functional association, genome context, and phyletic pattern. The results of classification and expert annotation are summarized in PIRSF family reports with graphical viewers for taxonomic distribution, domain architecture, family hierarchy, and multiple alignment and phylogenetic tree. The PIRSF system provides a comprehensive resource for bioinformatics analysis and comparative studies of protein function and evolution. Domain or fold-based searches allow identification of evolutionarily related protein families sharing domains or structural folds. Functional convergence and functional divergence are revealed by the relationships between protein classification and curated family functions. The taxonomic distribution allows the identification of lineage-specific or broadly conserved protein families and can reveal horizontal gene transfer. Here we demonstrate, with illustrative examples, how to use the web-based PIRSF system as a tool for functional and evolutionary studies of protein families.

  15. Intrachain disulfide bond in the core hinge region of human IgG4.

    PubMed Central

    Bloom, J. W.; Madanat, M. S.; Marriott, D.; Wong, T.; Chan, S. Y.

    1997-01-01

    IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than

  16. Maintenance of native-like protein dynamics may not be required for engineering functional proteins.

    PubMed

    Gobeil, Sophie M C; Clouthier, Christopher M; Park, Jaeok; Gagné, Donald; Berghuis, Albert M; Doucet, Nicolas; Pelletier, Joelle N

    2014-10-23

    Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.

  17. Understanding the Folding-Function Tradeoff in Proteins

    PubMed Central

    Gosavi, Shachi

    2013-01-01

    When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the “function-less” folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds. PMID:23593437

  18. Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

    PubMed

    Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-10-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

  19. Protein mechanics: a route from structure to function.

    PubMed

    Lavery, Richard; Sacquin-Mora, Sophie

    2007-08-01

    In order to better understand the mechanical properties of proteins, we have developed simulation tools which enable these properties to be analysed on a residue-by-residue basis. Although these calculations are relatively expensive with all-atom protein models, good results can be obtained much faster using coarse-grained approaches. The results show that proteins are surprisingly heterogeneous from a mechanical point of view and that functionally important residues often exhibit unusual mechanical behaviour. This finding offers a novel means for detecting functional sites and also potentially provides a route for understanding the links between structure and function in more general terms.

  20. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    PubMed Central

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-01-01

    Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices. PMID:26771082

  1. Function and structure of GFP-like proteins in the protein data bank.

    PubMed

    Ong, Wayne J-H; Alvarez, Samuel; Leroux, Ivan E; Shahid, Ramza S; Samma, Alex A; Peshkepija, Paola; Morgan, Alicia L; Mulcahy, Shawn; Zimmer, Marc

    2011-04-01

    The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.

  2. Accuracy of functional surfaces on comparatively modeled protein structures

    PubMed Central

    Zhao, Jieling; Dundas, Joe; Kachalo, Sema; Ouyang, Zheng; Liang, Jie

    2012-01-01

    Identification and characterization of protein functional surfaces are important for predicting protein function, understanding enzyme mechanism, and docking small compounds to proteins. As the rapid speed of accumulation of protein sequence information far exceeds that of structures, constructing accurate models of protein functional surfaces and identify their key elements become increasingly important. A promising approach is to build comparative models from sequences using known structural templates such as those obtained from structural genome projects. Here we assess how well this approach works in modeling binding surfaces. By systematically building three-dimensional comparative models of proteins using Modeller, we determine how well functional surfaces can be accurately reproduced. We use an alpha shape based pocket algorithm to compute all pockets on the modeled structures, and conduct a large-scale computation of similarity measurements (pocket RMSD and fraction of functional atoms captured) for 26,590 modeled enzyme protein structures. Overall, we find that when the sequence fragment of the binding surfaces has more than 45% identity to that of the tempalte protein, the modeled surfaces have on average an RMSD of 0.5 Å, and contain 48% or more of the binding surface atoms, with nearly all of the important atoms in the signatures of binding pockets captured. PMID:21541664

  3. Applications of Functional Protein Microarrays in Basic and Clinical Research

    PubMed Central

    Zhu, Heng; Qian, Jiang

    2013-01-01

    The protein microarray technology provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. It is viewed as a new tool that overcomes the limitation of DNA microarrays. On the basis of its application, protein microarrays fall into two major classes: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be directly spotted on a slide to form the so-called “reverse-phase” protein microarray. In the last decade, applications of functional protein microarrays in particular have flourished in studying protein function and construction of networks and pathways. In this chapter, we will review the recent advancements in the protein microarray technology, followed by presenting a series of examples to illustrate the power and versatility of protein microarrays in both basic and clinical research. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:22989767

  4. Targeting functional motifs of a protein family

    NASA Astrophysics Data System (ADS)

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  5. UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

    PubMed

    Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

    2016-01-01

    The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/.

  6. Elastic properties of protein functionalized nanoporous polymer films

    SciTech Connect

    Charles T. Black; Wang, Haoyu; Akcora, Pinar

    2015-12-16

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Lastly, our results show that protein functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces.

  7. Cellular strategies for regulating functional and nonfunctional protein aggregation.

    PubMed

    Gsponer, Jörg; Babu, M Madan

    2012-11-29

    Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier's principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

  8. 5. DETAIL OF HINGES ON RISING DOOR (OPEN) THAT REGULATES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. DETAIL OF HINGES ON RISING DOOR (OPEN) THAT REGULATES AIR FLOW - Sublet Mine No. 6, Fan House, North structure, west side of Willow Creek Valley, east of County Road No. 306, 3 miles north of U.S. Highway 189, Kemmerer, Lincoln County, WY

  9. Hinge specification for a square-faceted tetrahedral truss

    NASA Technical Reports Server (NTRS)

    Adams, L. R.

    1984-01-01

    A square-faceted tetrahedral truss is geometrically analyzed. Expressions are developed for single degree of freedom hinges which allow packaging of the structure into a configuration in which all members are parallel and closely packed in a square pattern. Deployment is sequential, thus providing control over the structure during deployment.

  10. Stiffness characterization of corner-filleted flexure hinges

    NASA Astrophysics Data System (ADS)

    Lobontiu, Nicolae; Garcia, Ephrahim; Hardau, Mihail; Bal, Nicolae

    2004-11-01

    The paper formulates the closed-form stiffness equations that can be used to characterize the static, modal, and dynamic behavior of single-axis corner-filleted flexure hinges, which are incorporated into macro/microscale monolithic mechanisms. The derivation is based on Castiliagno's first theorem and the resulting stiffness equations reflect sensitivity to direct- and cross-bending, axial loading, and torsion. Compared to previous analytical work, which assessed the stiffness of flexures by means of compliances, this paper directly gives the stiffness factors that completely define the elastic response of corner-filleted flexure hinges. The method is cost-effective as it requires considerably less calculation steps, compared to either finite element simulation or experimental characterization. Limit calculations demonstrate that the known engineering equations for a constant cross-section flexure are retrieved from those of a corner-filleted flexure hinge when the fillet radius becomes zero. The analytical model results are compared to experimental and finite element data and the errors are less than 8%. Further numerical simulation based on the analytical model highlights the influence of the geometric parameters on the stiffness properties of a corner-filleted flexure hinge.

  11. 61. DETAIL OF HEAD, VANE STEM, VANE HINGE, AND WHEEL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    61. DETAIL OF HEAD, VANE STEM, VANE HINGE, AND WHEEL OF AN ELI WINDMILL ON THE GROUND AT THE STOLL RESIDENCE ABOUT 1-1/2 MILES WEST OF NEBRASKA CITY ON STEAM WAGON ROAD. - Kregel Windmill Company Factory, 1416 Central Avenue, Nebraska City, Otoe County, NE

  12. Influence of the cardiac myosin hinge region on contractile activity.

    PubMed

    Margossian, S S; Krueger, J W; Sellers, J R; Cuda, G; Caulfield, J B; Norton, P; Slayter, H S

    1991-06-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

  13. A functional protein retention and release multilayer with high stability

    NASA Astrophysics Data System (ADS)

    Nie, Kun; An, Qi; Zhang, Yihe

    2016-04-01

    Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by varying the number of capping layers. Furthermore, we demonstrate that the protein-loaded interfacial layers could not only be used to construct catalytic-active interfaces, but also be integrated as the power-generating unit to propel a macroscopic floating device.Effective and robust interfacial protein retention lies at the heart of the fabrication of protein-based functional interfaces, which is potentially applicable in catalysis, medical therapy, antifouling, and smart devices, but remains challenging due to the sensitive nature of proteins. This study reports a general protein retention strategy to spatial-temporally confine various types of proteins at interfacial regions. The proteins were preserved in mesoporous silica nanoparticles embedded in covalently woven multilayers. It is worth noting that the protein retention strategy effectively preserves the catalytic capabilities of the proteins, and the multilayer structure is robust enough to withstand the bubbling catalytic reactions and could be repeatedly used due to conservation of proteins. The spatiotemporal retention of proteins could be adjusted by

  14. Regulation of bacterial RecA protein function.

    PubMed

    Cox, Michael M

    2007-01-01

    The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes. PMID:17364684

  15. Classifying proteins into functional groups based on all-versus-all BLAST of 10 million proteins.

    PubMed

    Kolker, Natali; Higdon, Roger; Broomall, William; Stanberry, Larissa; Welch, Dean; Lu, Wei; Haynes, Winston; Barga, Roger; Kolker, Eugene

    2011-01-01

    To address the monumental challenge of assigning function to millions of sequenced proteins, we completed the first of a kind all-versus-all sequence alignments using BLAST for 9.9 million proteins in the UniRef100 database. Microsoft Windows Azure produced over 3 billion filtered records in 6 days using 475 eight-core virtual machines. Protein classification into functional groups was then performed using Hive and custom jars implemented on top of Apache Hadoop utilizing the MapReduce paradigm. First, using the Clusters of Orthologous Genes (COG) database, a length normalized bit score (LNBS) was determined to be the best similarity measure for classification of proteins. LNBS achieved sensitivity and specificity of 98% each. Second, out of 5.1 million bacterial proteins, about two-thirds were assigned to significantly extended COG groups, encompassing 30 times more assigned proteins. Third, the remaining proteins were classified into protein functional groups using an innovative implementation of a single-linkage algorithm on an in-house Hadoop compute cluster. This implementation significantly reduces the run time for nonindexed queries and optimizes efficient clustering on a large scale. The performance was also verified on Amazon Elastic MapReduce. This clustering assigned nearly 2 million proteins to approximately half a million different functional groups. A similar approach was applied to classify 2.8 million eukaryotic sequences resulting in over 1 million proteins being assign to existing KOG groups and the remainder clustered into 100,000 functional groups. PMID:21809957

  16. Evaluation of epididymal function through specific protein on spermatozoa.

    PubMed

    Del Río, A G; De Sánchez, L Z; Sirena, A

    1984-01-01

    Investigations were focused on the characterization of specific epididymal proteins on the human spermatozoa as a representative parameter for epididymal function. An easy and attainable method, suitable for investigators and clinical use, is proposed in this article.

  17. Evaluation of epididymal function through specific protein on spermatozoa.

    PubMed

    Del Río, A G; De Sánchez, L Z; Sirena, A

    1984-01-01

    Investigations were focused on the characterization of specific epididymal proteins on the human spermatozoa as a representative parameter for epididymal function. An easy and attainable method, suitable for investigators and clinical use, is proposed in this article. PMID:6391400

  18. FunPred-1: protein function prediction from a protein interaction network using neighborhood analysis.

    PubMed

    Saha, Sovan; Chatterjee, Piyali; Basu, Subhadip; Kundu, Mahantapas; Nasipuri, Mita

    2014-12-01

    Proteins are responsible for all biological activities in living organisms. Thanks to genome sequencing projects, large amounts of DNA and protein sequence data are now available, but the biological functions of many proteins are still not annotated in most cases. The unknown function of such non-annotated proteins may be inferred or deduced from their neighbors in a protein interaction network. In this paper, we propose two new methods to predict protein functions based on network neighborhood properties. FunPred 1.1 uses a combination of three simple-yet-effective scoring techniques: the neighborhood ratio, the protein path connectivity and the relative functional similarity. FunPred 1.2 applies a heuristic approach using the edge clustering coefficient to reduce the search space by identifying densely connected neighborhood regions. The overall accuracy achieved in FunPred 1.2 over 8 functional groups involving hetero-interactions in 650 yeast proteins is around 87%, which is higher than the accuracy with FunPred 1.1. It is also higher than the accuracy of many of the state-of-the-art protein function prediction methods described in the literature. The test datasets and the complete source code of the developed software are now freely available at http://code.google.com/p/cmaterbioinfo/ . PMID:25424913

  19. FunPred-1: protein function prediction from a protein interaction network using neighborhood analysis.

    PubMed

    Saha, Sovan; Chatterjee, Piyali; Basu, Subhadip; Kundu, Mahantapas; Nasipuri, Mita

    2014-12-01

    Proteins are responsible for all biological activities in living organisms. Thanks to genome sequencing projects, large amounts of DNA and protein sequence data are now available, but the biological functions of many proteins are still not annotated in most cases. The unknown function of such non-annotated proteins may be inferred or deduced from their neighbors in a protein interaction network. In this paper, we propose two new methods to predict protein functions based on network neighborhood properties. FunPred 1.1 uses a combination of three simple-yet-effective scoring techniques: the neighborhood ratio, the protein path connectivity and the relative functional similarity. FunPred 1.2 applies a heuristic approach using the edge clustering coefficient to reduce the search space by identifying densely connected neighborhood regions. The overall accuracy achieved in FunPred 1.2 over 8 functional groups involving hetero-interactions in 650 yeast proteins is around 87%, which is higher than the accuracy with FunPred 1.1. It is also higher than the accuracy of many of the state-of-the-art protein function prediction methods described in the literature. The test datasets and the complete source code of the developed software are now freely available at http://code.google.com/p/cmaterbioinfo/ .

  20. Functions and mechanics of dynein motor proteins

    PubMed Central

    Roberts, Anthony J.; Kon, Takahide; Knight, Peter J.; Sutoh, Kazuo; Burgess, Stan A.

    2014-01-01

    Fuelled by ATP hydrolysis, dyneins generate force and movement on microtubules in a wealth of biological processes, including ciliary beating, cell division and intracellular transport. The large mass and complexity of dynein motors have made elucidating their mechanisms a sizable task. Yet, through a combination of approaches, including X-ray crystallography, cryo-electron microscopy, single-molecule assays and biochemical experiments, important progress has been made towards understanding how these giant motor proteins work. From these studies, a model for the mechanochemical cycle of dynein is emerging, in which nucleotide-driven flexing motions within the AAA+ ring of dynein alter the affinity of its microtubule-binding stalk and reshape its mechanical element to generate movement. PMID:24064538

  1. Tactile Teaching: Exploring Protein Structure/Function Using Physical Models

    ERIC Educational Resources Information Center

    Herman, Tim; Morris, Jennifer; Colton, Shannon; Batiza, Ann; Patrick, Michael; Franzen, Margaret; Goodsell, David S.

    2006-01-01

    The technology now exists to construct physical models of proteins based on atomic coordinates of solved structures. We review here our recent experiences in using physical models to teach concepts of protein structure and function at both the high school and the undergraduate levels. At the high school level, physical models are used in a…

  2. Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

    PubMed Central

    MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

    2008-01-01

    Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

  3. Discovering conformational sub-states relevant to protein function

    SciTech Connect

    Agarwal, Pratul K; Ramanathan, Arvind

    2011-01-01

    Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function.

  4. Assessment of protein set coherence using functional annotations

    PubMed Central

    Chagoyen, Monica; Carazo, Jose M; Pascual-Montano, Alberto

    2008-01-01

    Background Analysis of large-scale experimental datasets frequently produces one or more sets of proteins that are subsequently mined for functional interpretation and validation. To this end, a number of computational methods have been devised that rely on the analysis of functional annotations. Although current methods provide valuable information (e.g. significantly enriched annotations, pairwise functional similarities), they do not specifically measure the degree of homogeneity of a protein set. Results In this work we present a method that scores the degree of functional homogeneity, or coherence, of a set of proteins on the basis of the global similarity of their functional annotations. The method uses statistical hypothesis testing to assess the significance of the set in the context of the functional space of a reference set. As such, it can be used as a first step in the validation of sets expected to be homogeneous prior to further functional interpretation. Conclusion We evaluate our method by analysing known biologically relevant sets as well as random ones. The known relevant sets comprise macromolecular complexes, cellular components and pathways described for Saccharomyces cerevisiae, which are mostly significantly coherent. Finally, we illustrate the usefulness of our approach for validating 'functional modules' obtained from computational analysis of protein-protein interaction networks. Matlab code and supplementary data are available at PMID:18937846

  5. FUNCTIONAL INTERACTOMICS: DETERMINING THE ROLES PLAYED BY MEMBERS OF THE POPULAR BIOMASS PROTEIN-PROTEIN INTERACTOME

    SciTech Connect

    Beers, Eric; Brunner, Amy; Helm, Richard

    2015-07-31

    Proteins are molecular machines that are required for nearly all biological functions based on interactions with other molecules such as carbohydrates, lipids, other low molecular weight molecules, nucleic acids and other proteins. Here we map protein-protein interactions relevant to biomass production by focusing on proteins coexpressed in poplar xylem, the site of the majority of lignocellulose synthesis and hence biomass accumulation in poplar. Work proposed here will yield novel biological and bioinformatic resources that can benefit a variety of ongoing and future projects focusing on plant biomass/cell wall biology. The protein-protein interaction map that results from these studies will comprise an advanced view of protein-protein interactions in a model biomass tissue. Results will be made available to the biomass research community to serve as tools for developing new strategies for altering biomass quality and quantity.

  6. Automatic annotation of protein function based on family identification.

    PubMed

    Abascal, Federico; Valencia, Alfonso

    2003-11-15

    Although genomes are being sequenced at an impressive rate, the information generated tells us little about protein function, which is slow to characterize by traditional methods. Automatic protein function annotation based on computational methods has alleviated this imbalance. The most powerful current approach for inferring the function of new proteins is by studying the annotations of their homologues, since their common origin is assumed to be reflected in their structure and function. Unfortunately, as proteins evolve they acquire new functions, so annotation based on homology must be carried out in the context of orthologues or subfamilies. Evolution adds new complications through domain shuffling: homology (or orthology) frequently corresponds to domains rather than complete proteins. Moreover, the function of a protein may be seen as the result of combining the functions of its domains. Additionally, automatic annotation has to deal with problems related to the annotations in the databases: errors (which are likely to be propagated), inconsistencies, or different degrees of function specification. We describe a method that addresses these difficulties for the annotation of protein function. Sequence relationships are detected and measured to obtain a map of the sequence space, which is searched for differentiated groups of proteins (similar to islands on the map), which are expected to have a common function and correspond to groups of orthologues or subfamilies. This mapmaking is done by applying a clustering algorithm based on Normalized cuts in graphs. The domain problem is addressed in a simple way: pairwise local alignments are analyzed to determine the extent to which they cover the entire sequence lengths of the two proteins. This analysis determines both what homologues are preferred for functional inheritance and the level of confidence of the annotation. To alleviate the problems associated with database annotations, the information on all the

  7. CATH FunFHMMer web server: protein functional annotations using functional family assignments.

    PubMed

    Das, Sayoni; Sillitoe, Ian; Lee, David; Lees, Jonathan G; Dawson, Natalie L; Ward, John; Orengo, Christine A

    2015-07-01

    The widening function annotation gap in protein databases and the increasing number and diversity of the proteins being sequenced presents new challenges to protein function prediction methods. Multidomain proteins complicate the protein sequence-structure-function relationship further as new combinations of domains can expand the functional repertoire, creating new proteins and functions. Here, we present the FunFHMMer web server, which provides Gene Ontology (GO) annotations for query protein sequences based on the functional classification of the domain-based CATH-Gene3D resource. Our server also provides valuable information for the prediction of functional sites. The predictive power of FunFHMMer has been validated on a set of 95 proteins where FunFHMMer performs better than BLAST, Pfam and CDD. Recent validation by an independent international competition ranks FunFHMMer as one of the top function prediction methods in predicting GO annotations for both the Biological Process and Molecular Function Ontology. The FunFHMMer web server is available at http://www.cathdb.info/search/by_funfhmmer.

  8. CATH FunFHMMer web server: protein functional annotations using functional family assignments

    PubMed Central

    Das, Sayoni; Sillitoe, Ian; Lee, David; Lees, Jonathan G.; Dawson, Natalie L.; Ward, John; Orengo, Christine A.

    2015-01-01

    The widening function annotation gap in protein databases and the increasing number and diversity of the proteins being sequenced presents new challenges to protein function prediction methods. Multidomain proteins complicate the protein sequence–structure–function relationship further as new combinations of domains can expand the functional repertoire, creating new proteins and functions. Here, we present the FunFHMMer web server, which provides Gene Ontology (GO) annotations for query protein sequences based on the functional classification of the domain-based CATH-Gene3D resource. Our server also provides valuable information for the prediction of functional sites. The predictive power of FunFHMMer has been validated on a set of 95 proteins where FunFHMMer performs better than BLAST, Pfam and CDD. Recent validation by an independent international competition ranks FunFHMMer as one of the top function prediction methods in predicting GO annotations for both the Biological Process and Molecular Function Ontology. The FunFHMMer web server is available at http://www.cathdb.info/search/by_funfhmmer. PMID:25964299

  9. Functional assembly of protein fragments induced by spatial confinement.

    PubMed

    Yu, Yongsheng; Wang, Jianpeng; Liu, Jiahui; Ling, Daishun; Xia, Jiang

    2015-01-01

    Natural proteins are often confined within their local microenvironments, such as three-dimensional confinement in organelles or two-dimensional confinement in lipid rafts on cytoplasmic membrane. Spatial confinement restricts proteins' entropic freedom, forces their lateral interaction, and induces new properties that the same proteins lack at the soluble state. So far, the phenomenon of environment-induced protein functional alteration still lacks a full illustration. We demonstrate here that engineered protein fragments, although being non-functional in solution, can be re-assembled within the nanometer space to give the full activity of the whole protein. Specific interaction between hexahistidine-tag (His-tag) and NiO surface immobilizes protein fragments on NiO nanoparticles to form a self-assembled protein "corona" on the particles inside the nanopores of mesoporous silica. Site-specific assembly forces a shoulder-by-shoulder orientation and promotes fragment-fragment interaction; this interaction together with spatial confinement of the mesopores results in functional re-assembly of the protein half fragments. To our surprise, a single half fragment of luciferase (non-catalytic in solution) exhibited luciferase activity when immobilized on NiO in the mesopores, in the absence of the complimentary half. This shows for the first time that spatial confinement can induce the folding of a half fragment, reconstitute the enzyme active site, and re-gain the catalytic capability of the whole protein. Our work thereby highlights the under-documented notion that aside from the chemical composition such as primary sequence, physical environment of a protein also determines its function. PMID:25875003

  10. Functional anatomy of an allosteric protein

    NASA Astrophysics Data System (ADS)

    Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

    2013-12-01

    Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2-αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

  11. Function of platelet 47K protein phosphorylation

    SciTech Connect

    Imaoka, T.

    1987-05-01

    To provide insight into the biochemical pathway of platelet activation, they purified both unphosphorylated and phosphorylated P47 to homogeneity from human platelets. This study represents the first demonstration of a change of physiological action of P47 in response to phosphorylation in platelet activation. SVI labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presence of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had an inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8 (P47:actin). These activities were Ca independent. Purified TSP-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization. Therefore, they propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction.

  12. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  13. Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

    PubMed

    Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

    2013-03-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

  14. Dissecting Protein Function: An Efficient Protocol for Identifying Separation-of-Function Mutations That Encode Structurally Stable Proteins

    PubMed Central

    Lubin, Johnathan W.; Rao, Timsi; Mandell, Edward K.; Wuttke, Deborah S.; Lundblad, Victoria

    2013-01-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3− mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations. PMID:23307900

  15. Predicting protein functions from redundancies in large-scale protein interaction networks

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj Pratim; Liang, Shoudan

    2003-01-01

    Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives. Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data. Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations. Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein. By further analyzing these associations, we derive tentative functions for 81 unannotated proteins with high certainty. Our method is not overly sensitive to the false positives present in the data. Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (approximately 89%) of the original associations.

  16. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus. PMID:22428138

  17. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

  18. Artificial membranes for membrane protein purification, functionality and structure studies.

    PubMed

    Parmar, Mayuriben J; Lousa, Carine De Marcos; Muench, Stephen P; Goldman, Adrian; Postis, Vincent L G

    2016-06-15

    Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. PMID:27284055

  19. The APSES family proteins in fungi: Characterizations, evolution and functions.

    PubMed

    Zhao, Yong; Su, Hao; Zhou, Jing; Feng, Huihua; Zhang, Ke-Qin; Yang, Jinkui

    2015-08-01

    The APSES protein family belongs to transcriptional factors of the basic helix-loop-helix (bHLH) class, the originally described members (APSES: Asm1p, Phd1p, Sok2p, Efg1p and StuAp) are used to designate this group of proteins, and they have been identified as key regulators of fungal development and other biological processes. APSES proteins share a highly conserved DNA-binding domain (APSES domain) of about 100 amino acids, whose central domain is predicted to form a typical bHLH structure. Besides APSES domain, several APSES proteins also contain additional domains, such as KilA-N and ankyrin repeats. In recent years, an increasing number of APSES proteins have been identified from diverse fungi, and they involve in numerous biological processes, such as sporulation, cellular differentiation, mycelial growth, secondary metabolism and virulence. Most fungi, including Aspergillus fumigatus, Aspergillus nidulans, Candida albicans, Fusarium graminearum, and Neurospora crassa, contain five APSES proteins. However, Cryptococcus neoformans only contains two APSES proteins, and Saccharomyces cerevisiae contains six APSES proteins. The phylogenetic analysis showed the APSES domains from different fungi were grouped into four clades (A, B, C and D), which is consistent with the result of homologous alignment of APSES domains using DNAman. The roles of APSES proteins in clade C have been studied in detail, while little is known about the roles of other APSES proteins in clades A, B and D. In this review, the biochemical properties and functional domains of APSES proteins are predicted and compared, and the phylogenetic relationship among APSES proteins from various fungi are analyzed based on the APSES domains. Moreover, the functions of APSES proteins in different fungi are summarized and discussed.

  20. The APOBEC Protein Family: United by Structure, Divergent in Function.

    PubMed

    Salter, Jason D; Bennett, Ryan P; Smith, Harold C

    2016-07-01

    The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated. PMID:27283515

  1. Function of PPR proteins in plastid gene expression.

    PubMed

    Shikanai, Toshiharu; Fujii, Sota

    2013-01-01

    PPR proteins form a huge family in flowering plants and are involved in RNA maturation in plastids and mitochondria. These proteins are sequence-specific RNA-binding proteins that recruit the machinery of RNA processing. We summarize progress in the research on the functional mechanisms of divergent RNA maturation and on the mechanism by which RNA sequences are recognized. We further focus on two topics. RNA editing is an enigmatic process of RNA maturation in organelles, in which members of the PLS subfamily contribute to target site recognition. As the first topic, we speculate on why the PLS subfamily was selected by the RNA editing machinery. Second, we discuss how the regulation of plastid gene expression contributes to efficient photosynthesis. Although the molecular functions of PPR proteins have been studied extensively, information on the physiological significance of regulation by these proteins remains very limited.

  2. The APOBEC Protein Family: United by Structure, Divergent in Function.

    PubMed

    Salter, Jason D; Bennett, Ryan P; Smith, Harold C

    2016-07-01

    The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

  3. Recent advances in the photochemical control of protein function

    PubMed Central

    Riggsbee, Chad W.; Deiters, Alexander

    2010-01-01

    Biological processes are regulated with a high level of spatial and temporal resolution. In order to understand and manipulate these processes, scientists need to be able to regulate them with Nature’s level of precision. In this context, light is a unique regulatory element because it can be precisely controlled in location, timing and amplitude. Moreover, most biological laboratories have a wide range of light sources as standard equipment. This review article summarizes the most recent advances in light-mediated regulation of protein function and the application in a cellular context. Specifically, the photocaging of small molecule modulators of protein function and of select amino acid residues in proteins will be discussed. In addition, examples of the photochemical control of protein function through the application of natural light-receptors are presented. PMID:20667607

  4. Elastic properties of protein functionalized nanoporous polymer films

    DOE PAGES

    Charles T. Black; Wang, Haoyu; Akcora, Pinar

    2015-12-16

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Lastly, our results show that proteinmore » functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces.« less

  5. Determining protein function and interaction from genome analysis

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Thompson, Michael J.; Pellegrini, Matteo; Yeates, Todd O.

    2004-08-03

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  6. High Pressure NMR Methods for Characterizing Functional Substates of Proteins.

    PubMed

    Kalbitzer, Hans Robert

    2015-01-01

    Proteins usually exist in multiple conformational states in solution. High pressure NMR spectroscopy is a well-suited method to identify these states. In addition, these states can be characterized by their thermodynamic parameters, the free enthalpies at ambient pressure, the partial molar volumes, and the partial molar compressibility that can be obtained from the analysis of the high pressure NMR data. Two main types of states of proteins exist, functional states and folding states. There is a strong link between these two types, the functional states represent essential folding states (intermediates), other folding states may have no functional meaning (optional folding states). In this chapter, this concept is tested on the Ras protein, an important proto-oncogen in humans where all substates required by theory can be identified experimentally by high pressure NMR spectroscopy. Finally, we show how these data can be used to develop allosteric inhibitors of proteins. PMID:26174382

  7. The Protein Information Resource: an integrated public resource of functional annotation of proteins

    PubMed Central

    Wu, Cathy H.; Huang, Hongzhan; Arminski, Leslie; Castro-Alvear, Jorge; Chen, Yongxing; Hu, Zhang-Zhi; Ledley, Robert S.; Lewis, Kali C.; Mewes, Hans-Werner; Orcutt, Bruce C.; Suzek, Baris E.; Tsugita, Akira; Vinayaka, C. R.; Yeh, Lai-Su L.; Zhang, Jian; Barker, Winona C.

    2002-01-01

    The Protein Information Resource (PIR) serves as an integrated public resource of functional annotation of protein data to support genomic/proteomic research and scientific discovery. The PIR, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the PIR-International Protein Sequence Database (PSD), the major annotated protein sequence database in the public domain, containing about 250 000 proteins. To improve protein annotation and the coverage of experimentally validated data, a bibliography submission system is developed for scientists to submit, categorize and retrieve literature information. Comprehensive protein information is available from iProClass, which includes family classification at the superfamily, domain and motif levels, structural and functional features of proteins, as well as cross-references to over 40 biological databases. To provide timely and comprehensive protein data with source attribution, we have introduced a non-redundant reference protein database, PIR-NREF. The database consists of about 800 000 proteins collected from PIR-PSD, SWISS-PROT, TrEMBL, GenPept, RefSeq and PDB, with composite protein names and literature data. To promote database interoperability, we provide XML data distribution and open database schema, and adopt common ontologies. The PIR web site (http://pir.georgetown.edu/) features data mining and sequence analysis tools for information retrieval and functional identification of proteins based on both sequence and annotation information. The PIR databases and other files are also available by FTP (ftp://nbrfa.georgetown.edu/pir_databases). PMID:11752247

  8. Protein Conformational Populations and Functionally Relevant Sub-states

    SciTech Connect

    Agarwal, Pratul K; Burger, Virginia; Savol, Andrej; Ramanathan, Arvind; Chennubhotla, Chakra

    2013-01-01

    Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of the protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow

  9. Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

    PubMed Central

    Bloom, Jesse D; Romero, Philip A; Lu, Zhongyi; Arnold, Frances H

    2007-01-01

    Background Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein's ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, "promiscuous" functions that are incidental to a protein's original biological role. If mutations that are neutral with respect to a protein's primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution. Results Here we investigate this possibility experimentally by examining how cytochrome P450 enzymes that have evolved neutrally with respect to activity on a single substrate have changed in their abilities to catalyze reactions on five other substrates. We find that the enzymes have sometimes changed as much as four-fold in the promiscuous activities. The changes in promiscuous activities tend to increase with the number of mutations, and can be largely rationalized in terms of the chemical structures of the substrates. The activities on chemically similar substrates tend to change in a coordinated fashion, potentially providing a route for systematically predicting the change in one activity based on the measurement of several others. Conclusion Our work suggests that initially neutral genetic drift can lead to substantial changes in protein functions that are not currently under selection, in effect poising the proteins to more readily undergo functional evolution should selection favor new functions in the future. Reviewers This article was reviewed by Martijn Huynen, Fyodor Kondrashov, and Dan Tawfik (nominated by Christoph Adami). PMID:17598905

  10. Network Analysis of Circular Permutations in Multidomain Proteins Reveals Functional Linkages for Uncharacterized Proteins

    PubMed Central

    Adjeroh, Donald; Jiang, Yue; Jiang, Bing-Hua; Lin, Jie

    2014-01-01

    Various studies have implicated different multidomain proteins in cancer. However, there has been little or no detailed study on the role of circular multidomain proteins in the general problem of cancer or on specific cancer types. This work represents an initial attempt at investigating the potential for predicting linkages between known cancer-associated proteins with uncharacterized or hypothetical multidomain proteins, based primarily on circular permutation (CP) relationships. First, we propose an efficient algorithm for rapid identification of both exact and approximate CPs in multidomain proteins. Using the circular relations identified, we construct networks between multidomain proteins, based on which we perform functional annotation of multidomain proteins. We then extend the method to construct subnetworks for selected cancer subtypes, and performed prediction of potential link-ages between uncharacterized multidomain proteins and the selected cancer types. We include practical results showing the performance of the proposed methods. PMID:25741177

  11. Solid state protein monolayers: Morphological, conformational, and functional properties

    NASA Astrophysics Data System (ADS)

    Pompa, P. P.; Biasco, A.; Frascerra, V.; Calabi, F.; Cingolani, R.; Rinaldi, R.; Verbeet, M. Ph.; de Waal, E.; Canters, G. W.

    2004-12-01

    We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.

  12. How to Build a Complex, Functional Propeller Protein, From Parts.

    PubMed

    Clark, Patricia L

    2016-04-01

    By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed β-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo.

  13. Proteomics enhances evolutionary and functional analysis of reproductive proteins.

    PubMed

    Findlay, Geoffrey D; Swanson, Willie J

    2010-01-01

    Reproductive proteins maintain species-specific barriers to fertilization, affect the outcome of sperm competition, mediate reproductive conflicts between the sexes, and potentially contribute to the formation of new species. However, the specific proteins and molecular mechanisms that underlie these processes are understood in only a handful of cases. Advances in genomic and proteomic technologies enable the identification of large suites of reproductive proteins, making it possible to dissect reproductive phenotypes at the molecular level. We first review these technological advances and describe how reproductive proteins are identified in diverse animal taxa. We then discuss the dynamic evolution of reproductive proteins and the potential selective forces that act on them. Finally, we describe molecular and genomic tools for functional analysis and detail how evolutionary data may be used to make predictions about interactions among reproductive proteins.

  14. Structural and functional properties of hemp seed protein products.

    PubMed

    Malomo, Sunday A; He, Rong; Aluko, Rotimi E

    2014-08-01

    The effects of pH and protein concentration on some structural and functional properties of hemp seed protein isolate (HPI, 84.15% protein content) and defatted hemp seed protein meal (HPM, 44.32% protein content) were determined. The HPI had minimum protein solubility (PS) at pH 4.0, which increased as pH was decreased or increased. In contrast, the HPM had minimum PS at pH 3.0, which increased at higher pH values. Gel electrophoresis showed that some of the high molecular weight proteins (>45 kDa) present in HPM were not well extracted by the alkali and were absent or present in low ratio in the HPI polypeptide profile. The amino acid composition showed that the isolation process increased the Arg/Lys ratio of HPI (5.52%) when compared to HPM (3.35%). Intrinsic fluorescence and circular dichroism data indicate that the HPI proteins had a well-defined structure at pH 3.0, which was lost as pH value increased. The differences in structural conformation of HPI at different pH values were reflected as better foaming capacity at pH 3.0 when compared to pH 5.0, 7.0, and 9.0. At 10 and 25 mg/mL protein concentrations, emulsions formed by the HPM had smaller oil droplet sizes (higher quality), when compared to the HPI-formed emulsions. In contrast at 50 mg/mL protein concentration, the HPI-formed emulsions had smaller oil droplet sizes (except at pH 3.0). We conclude that the functional properties of hemp seed protein products are dependent on structural conformations as well as protein concentration and pH.

  15. Protein mechanics: from single molecules to functional biomaterials.

    PubMed

    Li, Hongbin; Cao, Yi

    2010-10-19

    Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of

  16. Functional annotation of hypothetical proteins – A review

    PubMed Central

    Sivashankari, Selvarajan; Shanmughavel, Piramanayagam

    2006-01-01

    The complete human genome sequences in the public database provide ways to understand the blue print of life. As of June 29, 2006, 27 archaeal, 326 bacterial and 21 eukaryotes is complete genomes are available and the sequencing for 316 bacterial, 24 archaeal, 126 eukaryotic genomes are in progress. The traditional biochemical/molecular experiments can assign accurate functions for genes in these genomes. However, the process is time-consuming and costly. Despite several efforts, only 50-60 % of genes have been annotated in most completely sequenced genomes. Automated genome sequence analysis and annotation may provide ways to understand genomes. Thus, determination of protein function is one of the challenging problems of the post-genome era. This demands bioinformatics to predict functions of un-annotated protein sequences by developing efficient tools. Here, we discuss some of the recent and popular approaches developed in Bioinformatics to predict functions for hypothetical proteins. PMID:17597916

  17. Functional Domain Motions in Proteins on the 1 100 ns Timescale: Comparison of Neutron Spin-Echo Spectroscopy of Phosphoglycerate Kinase with Molecular-Dynamics Simulation

    SciTech Connect

    Smolin, Nikolai; Biehl, R; Kneller, Gerald; Richter, Dieter O; Smith, Jeremy C

    2011-01-01

    Protein function often requires large-scale domain motion. An exciting new development in the experimental characterization of domain motions in proteins is the application of neutron spin-echo spectroscopy (NSE). NSE directly probes coherent (i.e., pair correlated) scattering on the 1 100 ns timescale. Here, we report on all-atom molecular-dynamics (MD) simulation of a protein, phosphoglycerate kinase, from which we calculate small-angle neutron scattering (SANS) and NSE scattering properties. The simulation-derived and experimental-solution SANS results are in excellent agreement. The contributions of translational and rotational whole-molecule diffusion to the simulation-derived NSE and potential problems in their estimation are examined. Principal component analysis identifies types of domain motion that dominate the internal motion's contribution to the NSE signal, with the largest being classic hinge bending. The associated free-energy profiles are quasiharmonic and the frictional properties correspond to highly overdamped motion. The amplitudes of the motions derived by MD are smaller than those derived from the experimental analysis, and possible reasons for this difference are discussed. The MD results confirm that a significant component of the NSE arises from internal dynamics. They also demonstrate that the combination of NSE with MD is potentially useful for determining the forms, potentials of mean force, and time dependence of functional domain motions in proteins.

  18. Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family.

    PubMed

    Thakur, Subarna; Bothra, Asim K; Sen, Arnab

    2013-11-01

    Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD and K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/ BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity. We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication. PMID:24287653

  19. Cellular functions of gamma-secretase-related proteins.

    PubMed

    Haffner, Christof; Haass, Christian

    2006-01-01

    Amyloid-beta peptide (Abeta) is generated by gamma-secretase, a membrane protein complex with an unusual aspartyl protease activity consisting of the four components presenilin, nicastrin, APH-1 and PEN-2. Presenilin is considered the catalytic subunit of this complex since it represents the prototype of the new family of intramembrane-cleaving GxGD-type aspartyl proteases. Recently, five novel members of this family and a nicastrin-like protein were identified. Whereas one of the GxGD-type proteins was shown to be identical with signal peptide peptidase (SPP), the function of the others, now called SPP-like proteins (SPPLs), is not known. We therefore analyzed SPPL2b and SPPL3 and demonstrated that they localize to different subcellular compartments suggesting nonredundant functions. This was supported by different phenotypes obtained in knockdown studies in zebrafish embryos. In addition, these phenotypes could be phenocopied by ectopic expression of putative active site mutants, providing strong evidence for a proteolytic function of SPPL2b and SPPL3. We also identified and characterized the nicastrin-like protein nicalin which, together with the 130-kDa protein NOMO (Nodal modulator), forms a membrane protein complex different from gamma-secretase. We found that during zebrafish embryogenesis this complex is involved in the patterning of the axial mesendoderm, a process controlled by the Nodal signaling pathway. PMID:17047369

  20. An Atomistic Statistically Effective Energy Function for Computational Protein Design.

    PubMed

    Topham, Christopher M; Barbe, Sophie; André, Isabelle

    2016-08-01

    Shortcomings in the definition of effective free-energy surfaces of proteins are recognized to be a major contributory factor responsible for the low success rates of existing automated methods for computational protein design (CPD). The formulation of an atomistic statistically effective energy function (SEEF) suitable for a wide range of CPD applications and its derivation from structural data extracted from protein domains and protein-ligand complexes are described here. The proposed energy function comprises nonlocal atom-based and local residue-based SEEFs, which are coupled using a novel atom connectivity number factor to scale short-range, pairwise, nonbonded atomic interaction energies and a surface-area-dependent cavity energy term. This energy function was used to derive additional SEEFs describing the unfolded-state ensemble of any given residue sequence based on computed average energies for partially or fully solvent-exposed fragments in regions of irregular structure in native proteins. Relative thermal stabilities of 97 T4 bacteriophage lysozyme mutants were predicted from calculated energy differences for folded and unfolded states with an average unsigned error (AUE) of 0.84 kcal mol(-1) when compared to experiment. To demonstrate the utility of the energy function for CPD, further validation was carried out in tests of its capacity to recover cognate protein sequences and to discriminate native and near-native protein folds, loop conformers, and small-molecule ligand binding poses from non-native benchmark decoys. Experimental ligand binding free energies for a diverse set of 80 protein complexes could be predicted with an AUE of 2.4 kcal mol(-1) using an additional energy term to account for the loss in ligand configurational entropy upon binding. The atomistic SEEF is expected to improve the accuracy of residue-based coarse-grained SEEFs currently used in CPD and to extend the range of applications of extant atom-based protein statistical

  1. An Atomistic Statistically Effective Energy Function for Computational Protein Design.

    PubMed

    Topham, Christopher M; Barbe, Sophie; André, Isabelle

    2016-08-01

    Shortcomings in the definition of effective free-energy surfaces of proteins are recognized to be a major contributory factor responsible for the low success rates of existing automated methods for computational protein design (CPD). The formulation of an atomistic statistically effective energy function (SEEF) suitable for a wide range of CPD applications and its derivation from structural data extracted from protein domains and protein-ligand complexes are described here. The proposed energy function comprises nonlocal atom-based and local residue-based SEEFs, which are coupled using a novel atom connectivity number factor to scale short-range, pairwise, nonbonded atomic interaction energies and a surface-area-dependent cavity energy term. This energy function was used to derive additional SEEFs describing the unfolded-state ensemble of any given residue sequence based on computed average energies for partially or fully solvent-exposed fragments in regions of irregular structure in native proteins. Relative thermal stabilities of 97 T4 bacteriophage lysozyme mutants were predicted from calculated energy differences for folded and unfolded states with an average unsigned error (AUE) of 0.84 kcal mol(-1) when compared to experiment. To demonstrate the utility of the energy function for CPD, further validation was carried out in tests of its capacity to recover cognate protein sequences and to discriminate native and near-native protein folds, loop conformers, and small-molecule ligand binding poses from non-native benchmark decoys. Experimental ligand binding free energies for a diverse set of 80 protein complexes could be predicted with an AUE of 2.4 kcal mol(-1) using an additional energy term to account for the loss in ligand configurational entropy upon binding. The atomistic SEEF is expected to improve the accuracy of residue-based coarse-grained SEEFs currently used in CPD and to extend the range of applications of extant atom-based protein statistical

  2. Phytochemicals perturb membranes and promiscuously alter protein function.

    PubMed

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  3. Computational approaches for inferring the functions of intrinsically disordered proteins

    PubMed Central

    Varadi, Mihaly; Vranken, Wim; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Intrinsically disordered proteins (IDPs) are ubiquitously involved in cellular processes and often implicated in human pathological conditions. The critical biological roles of these proteins, despite not adopting a well-defined fold, encouraged structural biologists to revisit their views on the protein structure-function paradigm. Unfortunately, investigating the characteristics and describing the structural behavior of IDPs is far from trivial, and inferring the function(s) of a disordered protein region remains a major challenge. Computational methods have proven particularly relevant for studying IDPs: on the sequence level their dependence on distinct characteristics determined by the local amino acid context makes sequence-based prediction algorithms viable and reliable tools for large scale analyses, while on the structure level the in silico integration of fundamentally different experimental data types is essential to describe the behavior of a flexible protein chain. Here, we offer an overview of the latest developments and computational techniques that aim to uncover how protein function is connected to intrinsic disorder. PMID:26301226

  4. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  5. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  6. Metrnl: a secreted protein with new emerging functions

    PubMed Central

    Zheng, Si-li; Li, Zhi-yong; Song, Jie; Liu, Jian-min; Miao, Chao-yu

    2016-01-01

    Secreted proteins play critical roles in physiological and pathological processes and can be used as biomarkers and therapies for aging and disease. Metrnl is a novel secreted protein homologous to the neurotrophin Metrn. But this protein, unlike Metrn that is mainly expressed in the brain, shows a relatively wider distribution in the body with high levels of expression in white adipose tissue and barrier tissues. This protein plays important roles in neural development, white adipose browning and insulin sensitization. Based on its expression and distinct functions, this protein is also called Cometin, Subfatin and Interleukin 39, which refer to its neurotrophic effect, adipokine function and the possible action as a cytokine, respectively. The spectrum of Metrnl functions remains to be determined, and the mechanisms of Metrnl action need to be elucidated. In this review, we focus on the discovery, structural characteristics, expression pattern and physiological functions of Metrnl, which will assist in developing this protein as a new therapeutic target or agent. PMID:27063217

  7. Gene3D: modelling protein structure, function and evolution.

    PubMed

    Yeats, Corin; Maibaum, Michael; Marsden, Russell; Dibley, Mark; Lee, David; Addou, Sarah; Orengo, Christine A

    2006-01-01

    The Gene3D release 4 database and web portal (http://cathwww.biochem.ucl.ac.uk:8080/Gene3D) provide a combined structural, functional and evolutionary view of the protein world. It is focussed on providing structural annotation for protein sequences without structural representatives--including the complete proteome sets of over 240 different species. The protein sequences have also been clustered into whole-chain families so as to aid functional prediction. The structural annotation is generated using HMM models based on the CATH domain families; CATH is a repository for manually deduced protein domains. Amongst the changes from the last publication are: the addition of over 100 genomes and the UniProt sequence database, domain data from Pfam, metabolic pathway and functional data from COGs, KEGG and GO, and protein-protein interaction data from MINT and BIND. The website has been rebuilt to allow more sophisticated querying and the data returned is presented in a clearer format with greater functionality. Furthermore, all data can be downloaded in a simple XML format, allowing users to carry out complex investigations at their own computers.

  8. Approaches for functional analysis of flagellar proteins in African trypanosomes.

    PubMed

    Oberholzer, Michael; Lopez, Miguel A; Ralston, Katherine S; Hill, Kent L

    2009-01-01

    The eukaryotic flagellum is a highly conserved organelle serving motility, sensory, and transport functions. Although genetic, genomic, and proteomic studies have led to the identification of hundreds of flagellar and putative flagellar proteins, precisely how these proteins function individually and collectively to drive flagellum motility and other functions remains to be determined. In this chapter we provide an overview of tools and approaches available for studying flagellum protein function in the protozoan parasite Trypanosoma brucei. We begin by outlining techniques for in vitro cultivation of both T. brucei life cycle stages, as well as transfection protocols for the delivery of DNA constructs. We then describe specific assays used to assess flagellum function including flagellum preparation and quantitative motility assays. We conclude the chapter with a description of molecular genetic approaches for manipulating gene function. In summary, the availability of potent molecular tools, as well as the health and economic relevance of T. brucei as a pathogen, combine to make the parasite an attractive and integral experimental system for the functional analysis of flagellar proteins. PMID:20409810

  9. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications

    PubMed Central

    Nader, Nancy; Chrousos, George P.; Kino, Tomoshige

    2009-01-01

    Glucocorticoids, end products of the hypothalamic-pituitary-adrenal axis, influence functions of virtually all organs and tissues through the glucocorticoid receptor (GR). Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms in both the central nervous system and periphery. We found that CLOCK/BMAL1 repressed GR-induced transcriptional activity in a HAT-activity- dependent fashion. In serum-shock-synchronized cells, transactivational activity of GR, accessed by mRNA expression of an endogenous-responsive gene, fluctuated spontaneously in a circadian fashion in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically, and CLOCK suppressed binding of GR to its DNA recognition sequences by acetylating multiple lysine residues located in its hinge region. These findings indicate that CLOCK/BMAL1 functions as a reverse-phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing biological actions of diurnally fluctuating circulating glucocorticoids. Further, these results suggest that a peripheral target tissue circadian rhythm indirectly influences the functions of every organ and tissue inside the body through modulation of the ubiquitous and diverse actions of glucocorticoids.—Nader, N., Chrousos, G. P., Kino, T. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications. PMID:19141540

  10. The regulation and function of the Id proteins in lymphocyte development.

    PubMed

    Rivera, R; Murre, C

    2001-12-20

    Helix-loop-helix (HLH) proteins are essential factors for lymphocyte development and function. One class of HLH proteins, the E-proteins, regulate many aspects of lymphocyte maturation, survival, proliferation, and differentiation. E-proteins are negatively regulated by another class of HLH proteins known as the Id proteins. The Id proteins function as dominant negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here we discuss the function and regulation of the Id proteins in lymphocyte development.

  11. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  12. Functional classification of CATH superfamilies: a domain-based approach for protein function annotation

    PubMed Central

    Das, Sayoni; Lee, David; Sillitoe, Ian; Dawson, Natalie L.; Lees, Jonathan G.; Orengo, Christine A.

    2015-01-01

    Motivation: Computational approaches that can predict protein functions are essential to bridge the widening function annotation gap especially since <1.0% of all proteins in UniProtKB have been experimentally characterized. We present a domain-based method for protein function classification and prediction of functional sites that exploits functional sub-classification of CATH superfamilies. The superfamilies are sub-classified into functional families (FunFams) using a hierarchical clustering algorithm supervised by a new classification method, FunFHMMer. Results: FunFHMMer generates more functionally coherent groupings of protein sequences than other domain-based protein classifications. This has been validated using known functional information. The conserved positions predicted by the FunFams are also found to be enriched in known functional residues. Moreover, the functional annotations provided by the FunFams are found to be more precise than other domain-based resources. FunFHMMer currently identifies 110 439 FunFams in 2735 superfamilies which can be used to functionally annotate > 16 million domain sequences. Availability and implementation: All FunFam annotation data are made available through the CATH webpages (http://www.cathdb.info). The FunFHMMer webserver (http://www.cathdb.info/search/by_funfhmmer) allows users to submit query sequences for assignment to a CATH FunFam. Contact: sayoni.das.12@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26139634

  13. A General Method for Insertion of Functional Proteins within Proteins via Combinatorial Selection of Permissive Junctions.

    PubMed

    Peng, Yingjie; Zeng, Wenwen; Ye, Hui; Han, Kyung Ho; Dharmarajan, Venkatasubramanian; Novick, Scott; Wilson, Ian A; Griffin, Patrick R; Friedman, Jeffrey M; Lerner, Richard A

    2015-08-20

    A major goal of modern protein chemistry is to create new proteins with different functions. One approach is to amalgamate secondary and tertiary structures from different proteins. This is difficult for several reasons, not the least of which is the fact that the junctions between secondary and tertiary structures are not degenerate and usually affect the function and folding of the entire complex. Here, we offer a solution to this problem by coupling a large combinatorial library of about 10(7) different N- and C-terminal junctions to a powerful system that selects for function. Using this approach, the entire Leptin and follicle-stimulating hormone (FSH) were inserted into an antibody. Complexes with full retention of function in vivo and in vitro, although rare, were found easily by using an autocrine selection system to search for hormonal activity. Such large diversity systems, when coupled to robust selection systems, should enable construction of novel therapeutic proteins.

  14. High-resolution mapping of protein sequence-function relationships.

    PubMed

    Fowler, Douglas M; Araya, Carlos L; Fleishman, Sarel J; Kellogg, Elizabeth H; Stephany, Jason J; Baker, David; Fields, Stanley

    2010-09-01

    We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

  15. A stepwise approach for the management of capsular contraction syndrome in hinge-based accommodative intraocular lenses

    PubMed Central

    Page, Timothy P; Whitman, Jeffrey

    2016-01-01

    Purpose The aims of this study are to define the various stages of capsular contraction syndrome (CCS) and its effect on refractive error with hinge-based accommodating intraocular lenses (IOLs) and to describe a systematic approach for the management of the different stages of CCS. Methods Hinge-based accommodative IOLs function via flexible hinges that vault the optic forward during accommodation. However, it is the flexibility of the IOL that makes it prone to deformation in the event of CCS. The signs of CCS are identified and described as posterior capsular striae, fibrotic bands across the anterior or posterior capsule, and capsule opacification. Various degrees of CCS may affect hinge-based accommodating IOLs in a spectrum from subtle changes in IOL appearance to significant increases in refractive error and loss of uncorrected visual acuity. The signs of CCS and its effect on IOL position and the resulting changes in refractive error are matched to appropriate treatment plans. Results A surgeon can avoid CCS and manage the condition if familiar with the early signs of CCS. If CCS is identified, yttrium–aluminum–garnet laser capsulotomy should be considered. If moderate CCS occurs, it may be effectively treated with insertion of a capsular tension ring. If CCS is allowed to progress to advanced stages, an IOL exchange may be necessary. Conclusion Surgeons should be familiar with the stages of CCS and subsequent interventions. The steps outlined in this article help to guide surgeons in the prevention and management of CCS with hinge-based accommodative IOLs in order to provide improved refractive outcomes for patients. PMID:27354757

  16. Optimizing high performance computing workflow for protein functional annotation.

    PubMed

    Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

    2014-09-10

    Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data. PMID:25313296

  17. Optimizing high performance computing workflow for protein functional annotation.

    PubMed

    Stanberry, Larissa; Rekepalli, Bhanu; Liu, Yuan; Giblock, Paul; Higdon, Roger; Montague, Elizabeth; Broomall, William; Kolker, Natali; Kolker, Eugene

    2014-09-10

    Functional annotation of newly sequenced genomes is one of the major challenges in modern biology. With modern sequencing technologies, the protein sequence universe is rapidly expanding. Newly sequenced bacterial genomes alone contain over 7.5 million proteins. The rate of data generation has far surpassed that of protein annotation. The volume of protein data makes manual curation infeasible, whereas a high compute cost limits the utility of existing automated approaches. In this work, we present an improved and optmized automated workflow to enable large-scale protein annotation. The workflow uses high performance computing architectures and a low complexity classification algorithm to assign proteins into existing clusters of orthologous groups of proteins. On the basis of the Position-Specific Iterative Basic Local Alignment Search Tool the algorithm ensures at least 80% specificity and sensitivity of the resulting classifications. The workflow utilizes highly scalable parallel applications for classification and sequence alignment. Using Extreme Science and Engineering Discovery Environment supercomputers, the workflow processed 1,200,000 newly sequenced bacterial proteins. With the rapid expansion of the protein sequence universe, the proposed workflow will enable scientists to annotate big genome data.

  18. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties.

    PubMed

    Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P; Landberg, Rikard; Langton, Maud

    2016-01-01

    A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications.

  19. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties

    PubMed Central

    Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P.; Landberg, Rikard; Langton, Maud

    2016-01-01

    A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications. PMID:26840533

  20. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties.

    PubMed

    Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P; Landberg, Rikard; Langton, Maud

    2016-01-01

    A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications. PMID:26840533

  1. KNEE ARTHROPLASTY REVISION WITH A CONSTRAINED IMPLANT USING HINGE AND ROTATING TIBIAL BASIS

    PubMed Central

    Angelini, Fabio Jansen; Helito, Camilo Partezani; Veronesi, Bruno Azevedo; Guimarães, Tales Mollica; Pécora, José Ricardo; Demange, Marco Kawamura

    2016-01-01

    ABSTRACT Objective: To evaluate the results of total knee arthoplasty revisions performed in high complexity cases, with large bone defects or serious ligament deficiencies using a constrained implant hinge associated to a rotating tibial basis. Methods: We evaluated 11 patients in which we used the constrained implant hinge associated to rotating tibial basis, with minimum follow-up of two years. The indications for the procedure included instability, septic loosening, late postoperative infection without loosening and periprosthetic fracture. We evaluated the knee range of movement and functional outcomes by the Knee Society Score (KSS) e Knee Injury and Osteoarthritis Outcome Score (KOOS), besides the presence of complications. Results: All patients achieved 5o to 85o minimum range of motion at 1 year postoperatively and, in the present evaluation, KSS ranged from 67 to 95. Three patients had no complications until the last evaluation and two patients required implant revision. Conclusion: Despite the complications rate observed, the functional result were acceptable for most patients, and it proved being a viable alternative, especially for patients with low functional demand. Level of Evidence IV, Case Series. PMID:26997909

  2. Versatile multi-functionalization of protein nanofibrils for biosensor applications

    NASA Astrophysics Data System (ADS)

    Sasso, L.; Suei, S.; Domigan, L.; Healy, J.; Nock, V.; Williams, M. A. K.; Gerrard, J. A.

    2014-01-01

    Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the

  3. Specific protein homeostatic functions of small heat-shock proteins increase lifespan.

    PubMed

    Vos, Michel J; Carra, Serena; Kanon, Bart; Bosveld, Floris; Klauke, Karin; Sibon, Ody C M; Kampinga, Harm H

    2016-04-01

    During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.

  4. Influence of the Cardiac Myosin Hinge Region on Contractile Activity

    NASA Astrophysics Data System (ADS)

    Margossian, Sarkis S.; Krueger, John W.; Sellers, James R.; Cuda, Giovanni; Caulfield, James B.; Norton, Paul; Slayter, Henry S.

    1991-06-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the antihinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

  5. Biomechanical Analysis of the Effects of Bilateral Hinged Knee Bracing

    PubMed Central

    Lee, Hangil; Ha, Dokyeong; Kang, Yeoun-Seung; Park, Hyung-Soon

    2016-01-01

    This research analyzed the effect of bilateral hinged knee braces on a healthy knee from a biomechanical frame in vivo. This was accomplished by fitting a knee brace with two customized wireless force/torque (F/T) sensors that could readily record force and torque during live motion, while the kinetics at the knee were computed using the inverse dynamics of the motion capture and force plate data. Four tasks to test the brace’s effects were drop vertical jumping, pivoting, stop vertical jumping, and cutting. The results showed that the hinges in the knee brace can absorb up to 18% of the force and 2.7% of the torque at the knee during various athletic motions. Thus, the hinges demonstrated minimal effect in reducing the mechanical load on the knee. There were limitations concerning the consistency of the motions performed by the subjects during the trials and the influence of the other portions of the brace to evaluate the overall effectiveness of the brace as a whole. Future works may incorporate a fatigue protocol and injured subjects to better determine the effects of the brace. There is still a need for more research on the biomechanical influence of knee braces to develop safer and more effective products. PMID:27379233

  6. Functional Characteristics of Milk Protein Concentrates and Their Modification.

    PubMed

    Uluko, Hankie; Liu, Lu; Lv, Jia-Ping; Zhang, Shu-Wen

    2016-05-18

    A major deterrent to the usage of milk protein concentrate (MPC), a high-protein milk product with increasing demand as a food and sports drink ingredient, has been its poor functional characteristics when compared with other milk protein products such as whey protein concentrate and sodium caseinates. This review discusses the recent research on functional properties of MPC, focusing on factors that may contribute to the poor functional characteristics before, during, and after production. Current research, methods employed, and new understanding on the causes of poor solubility of MPC at mild temperatures (about 20°C) has been presented, including loss of solubility during storage as these areas have received unprecedented attention over the past decade, and also affects other useful functional properties of MPC, such as emulsifying properties, gelation, and foaming. Processing methods, which include heat treatment, high-pressure application, microwave heating, ultrasound application, and enzyme and salts modification, have been used or have potential to modify or improve the functional properties of MPCs. Future research on the effects of these processing methods on the functional properties, including effects of enzyme hydrolysis on bitterness and bioactivity, has also been discussed. PMID:26048645

  7. Functional Characteristics of Milk Protein Concentrates and Their Modification.

    PubMed

    Uluko, Hankie; Liu, Lu; Lv, Jia-Ping; Zhang, Shu-Wen

    2016-05-18

    A major deterrent to the usage of milk protein concentrate (MPC), a high-protein milk product with increasing demand as a food and sports drink ingredient, has been its poor functional characteristics when compared with other milk protein products such as whey protein concentrate and sodium caseinates. This review discusses the recent research on functional properties of MPC, focusing on factors that may contribute to the poor functional characteristics before, during, and after production. Current research, methods employed, and new understanding on the causes of poor solubility of MPC at mild temperatures (about 20°C) has been presented, including loss of solubility during storage as these areas have received unprecedented attention over the past decade, and also affects other useful functional properties of MPC, such as emulsifying properties, gelation, and foaming. Processing methods, which include heat treatment, high-pressure application, microwave heating, ultrasound application, and enzyme and salts modification, have been used or have potential to modify or improve the functional properties of MPCs. Future research on the effects of these processing methods on the functional properties, including effects of enzyme hydrolysis on bitterness and bioactivity, has also been discussed.

  8. Twenty years of protein interaction studies for biological function deciphering.

    PubMed

    Legrain, Pierre; Rain, Jean-Christophe

    2014-07-31

    Intensive methodological developments and technology innovation have been devoted to protein-protein interaction studies over 20years. Genetic indirect assays and sophisticated large scale biochemical analyses have jointly contributed to the elucidation of protein-protein interactions, still with a lot of drawbacks despite heavy investment in human resources and technologies. With the most recent developments in mass spectrometry and computational tools for studying protein content of complex samples, the initial goal of deciphering molecular bases of biological functions is now within reach. Here, we described the various steps of this process and gave examples of key milestones in this scientific story line. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

  9. Functions and Regulation of the APOBEC Family of Proteins

    PubMed Central

    Smith, Harold C.; Bennett, Ryan P.; Kizilyer, Ayse; McDougall, William M.; Prohaska, Kimberly M.

    2012-01-01

    APOBEC1 is a cytidine deaminase that edits messenger RNAs and was the first enzyme in the APOBEC family to be functionally characterized. Under appropriate conditions APOBEC1 also deaminates deoxycytidine in single-stranded DNA (ssDNA). The other ten members of the APOBEC family have not been fully characterized however several have deoxycytidine deaminase activity on ssDNAs. Despite the nucleic acid substrate preferences of different APOBEC proteins, a common feature appears to be their intrinsic ability to bind to RNA as well as to ssDNA. RNA binding to APOBEC proteins together with protein-protein interactions, post-translation modifications and subcellular localization serve as biological modulators controlling the DNA mutagenic activity of these potentially genotoxic proteins. PMID:22001110

  10. The changing landscape in translocator protein (TSPO) function.

    PubMed

    Selvaraj, Vimal; Stocco, Douglas M

    2015-07-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is an outer mitochondrial membrane protein. TSPO has been shown to cooperate with steroidogenic acute regulatory protein (StAR) and function in the transport of cholesterol into mitochondria. TSPO has also been considered as a structural component of the mitochondrial permeability transition pore (MPTP). However, recent advances have changed these views of TSPO's functions and have prompted a re-evaluation of established concepts. This review summarizes the history of TSPO, key elements of the debate, and functional experiments that have changed our understanding. Moving forward, we examine how this fundamental change impacts our understanding of TSPO and affects the future of TSPO as a therapeutic and diagnostic target.

  11. Role of AAA(+)-proteins in peroxisome biogenesis and function.

    PubMed

    Grimm, Immanuel; Erdmann, Ralf; Girzalsky, Wolfgang

    2016-05-01

    Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function.

  12. Proteins that associate with lamins: Many faces, many functions

    SciTech Connect

    Schirmer, Eric C. . E-mail: e.schirmer@ed.ac.uk; Foisner, Roland . E-mail: roland.foisner@meduniwien.ac.at

    2007-06-10

    Lamin-associated polypeptides (LAPs) comprise inner nuclear membrane proteins tightly associated with the peripheral lamin scaffold as well as proteins forming stable complexes with lamins in the nucleoplasm. The involvement of LAPs in a wide range of human diseases may be linked to an equally bewildering range of their functions, including sterol reduction, histone modification, transcriptional repression, and Smad- and {beta}-catenin signaling. Many LAPs are likely to be at the center of large multi-protein complexes, components of which may dictate their functions, and a few LAPs have defined enzymatic activities. Here we discuss the definition of LAPs, review their many binding partners, elaborate their functions in nuclear architecture, chromatin organization, gene expression and signaling, and describe what is currently known about their links to human disease.

  13. Pattern recognition methods for protein functional site prediction.

    PubMed

    Yang, Zheng Rong; Wang, Lipo; Young, Natasha; Trudgian, Dave; Chou, Kuo-Chen

    2005-10-01

    Protein functional site prediction is closely related to drug design, hence to public health. In order to save the cost and the time spent on identifying the functional sites in sequenced proteins in biology laboratory, computer programs have been widely used for decades. Many of them are implemented using the state-of-the-art pattern recognition algorithms, including decision trees, neural networks and support vector machines. Although the success of this effort has been obvious, advanced and new algorithms are still under development for addressing some difficult issues. This review will go through the major stages in developing pattern recognition algorithms for protein functional site prediction and outline the future research directions in this important area. PMID:16248799

  14. Functional analysis of glucan binding protein B from Streptococcus mutans.

    PubMed

    Mattos-Graner, Renata O; Porter, Kristen A; Smith, Daniel J; Hosogi, Yumiko; Duncan, Margaret J

    2006-06-01

    Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division. PMID:16707674

  15. Electron crystallography for structural and functional studies of membrane proteins.

    PubMed

    Fujiyoshi, Yoshinori

    2011-01-01

    Membrane proteins are important research targets for basic biological sciences and drug design, but studies of their structure and function are considered difficult to perform. Studies of membrane structures have been greatly facilitated by technological and instrumental advancements in electron microscopy together with methodological advancements in biology. Electron crystallography is especially useful in studying the structure and function of membrane proteins. Electron crystallography is now an established method of analyzing the structures of membrane proteins in lipid bilayers, which resembles their natural biological environment. To better understand the neural system function from a structural point of view, we developed the cryo-electron microscope with a helium-cooled specimen stage, which allows for analysis of the structures of membrane proteins at a resolution higher than 3 Å. This review introduces recent instrumental advances in cryo-electron microscopy and presents some examples of structure analyses of membrane proteins, such as bacteriorhodopsin, water channels and gap junction channels. This review has two objectives: first, to provide a personal historical background to describe how we came to develop the cryo-electron microscope and second, to discuss some of the technology required for the structural analysis of membrane proteins based on cryo-electron microscopy.

  16. Evolutionary perspectives on protein structure, stability, and functionality

    NASA Astrophysics Data System (ADS)

    Goldstein, Richard A.

    Proteins are the result of a long process of evolution. It is due to this process that they have developed properties rather different from those of random strings of amino acids. If we wish to understand the properties of proteins, we need to understand the underlying process of Darwinian evolution, and how its stochastic nature interacts with the underlying fitness landscape. In this review, I describe some of the underlying theory of evolution. I then discuss how these theories can help us understand the structure, thermodynamics, and functioning of naturally-occurring proteins.

  17. Structures and functions of autotransporter proteins in microbial pathogens.

    PubMed

    Benz, Inga; Schmidt, M Alexander

    2011-08-01

    Since their discovery more than 20 years ago the autotransporter protein superfamily has been growing continuously and currently represents the largest protein family in (pathogenic) Gram-negative bacteria. Autotransporter proteins (AT) adhere to a common structural principle and are composed of a C-terminal β-barrel-shaped 'translocator' domain and an N-terminal 'passenger' domain. The translocator is anchored in the outer membrane and is indispensable for the N-terminal passenger part to traverse the outer membrane. Most if not all AT harbor a chaperone segment that increases protein stability and may be located in the passenger or translocator domain. The passenger mediates the specific virulence function(s) of the particular AT. Accordingly, passenger domains of AT can be quite variable. Interestingly, AT have been identified as the first glycosylated proteins in Gram-negative bacteria. Despite the considerable efforts invested in the characterization of autotransporter biogenesis, various aspects such as the participation of accessory proteins, the fate of the translocator, or the translocation of glycosylated proteins still remain only poorly understood. In addition, recent evidence indicates that the prefix 'auto' might be slightly exaggerated. Here, we will selectively discuss novel insights at various stages of AT biogenesis. PMID:21616712

  18. Functional conservation of an ancestral Pellino protein in helminth species.

    PubMed

    Cluxton, Christopher D; Caffrey, Brian E; Kinsella, Gemma K; Moynagh, Paul N; Fares, Mario A; Fallon, Padraic G

    2015-01-01

    The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans. PMID:26120048

  19. Functional conservation of an ancestral Pellino protein in helminth species.

    PubMed

    Cluxton, Christopher D; Caffrey, Brian E; Kinsella, Gemma K; Moynagh, Paul N; Fares, Mario A; Fallon, Padraic G

    2015-01-01

    The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans.

  20. Functional conservation of an ancestral Pellino protein in helminth species

    PubMed Central

    Cluxton, Christopher D.; Caffrey, Brian E.; Kinsella, Gemma K.; Moynagh, Paul N.; Fares, Mario A.; Fallon, Padraic G.

    2015-01-01

    The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans. PMID:26120048

  1. Functionality of alternative protein in gluten-free product development.

    PubMed

    Deora, Navneet Singh; Deswal, Aastha; Mishra, Hari Niwas

    2015-07-01

    Celiac disease is an immune-mediated disease triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. The current treatment for celiac disease is life-long adherence to a strict gluten-exclusion diet. The replacement of gluten presents a significant technological challenge, as it is an essential structure-building protein, which is necessary for formulating high-quality baked goods. A major limitation in the production of gluten-free products is the lack of protein functionality in non-wheat cereals. Additionally, commercial gluten-free mixes usually contain only carbohydrates, which may significantly limit the amount of protein in the diet. In the recent past, various approaches are attempted to incorporate protein-based ingredients and to modify the functional properties for gluten-free product development. This review aims to the highlight functionality of the alternative protein-based ingredients, which can be utilized for gluten-free product development both functionally as well as nutritionally.

  2. Functionality of alternative protein in gluten-free product development.

    PubMed

    Deora, Navneet Singh; Deswal, Aastha; Mishra, Hari Niwas

    2015-07-01

    Celiac disease is an immune-mediated disease triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. The current treatment for celiac disease is life-long adherence to a strict gluten-exclusion diet. The replacement of gluten presents a significant technological challenge, as it is an essential structure-building protein, which is necessary for formulating high-quality baked goods. A major limitation in the production of gluten-free products is the lack of protein functionality in non-wheat cereals. Additionally, commercial gluten-free mixes usually contain only carbohydrates, which may significantly limit the amount of protein in the diet. In the recent past, various approaches are attempted to incorporate protein-based ingredients and to modify the functional properties for gluten-free product development. This review aims to the highlight functionality of the alternative protein-based ingredients, which can be utilized for gluten-free product development both functionally as well as nutritionally. PMID:26048849

  3. Structural and functional analysis of fatty acid-binding proteins

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

    The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis. PMID:19017610

  4. [Functionally-relevant conformational dynamics of water-soluble proteins].

    PubMed

    Novikov, G V; Sivozhelezov, V S; Shaĭtan, K V

    2013-01-01

    A study is reported of the functional-relevant dynamics of three typical water-soluble proteins: Calmodulin, Src-tyrosine kinase as well as repressor of Trp operon. Application of the state-of-art methods of structural bioinformatics allowed to identify dynamics seen in the X-ray structures of the investigated proteins associated with their specific biological functions. In addition, Normal Mode analysis technique revealed the most probable directions of the functionally-relevant motions for all that proteins were also predicted. Importantly, overall type of the motions observed on the lowest-frequency modes was very similar to the motions seen from the analysis of the X-ray data of the examined macromolecules. Thereby it was shown that the large-scale as well as local conformational motions of the proteins might be predetermined already at the level of their tertiary structures. In particular, the determining factor might be the specific fold of the alpha-helixes. Thus functionally-relevant in vivo dynamics of the investigated proteins might be evolutionally formed by means of natural selection at the level of the spatial topology. PMID:23705506

  5. Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

    PubMed

    Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T; Minogue, Catie E; Xia, Chuanwu; Beebe, Emily T; Wrobel, Russell L; Cho, Holly; Kremer, Laura S; Alston, Charlotte L; Gromek, Katarzyna A; Dolan, Brendan K; Ulbrich, Arne; Stefely, Jonathan A; Bohl, Sarah L; Werner, Kelly M; Jochem, Adam; Westphall, Michael S; Rensvold, Jarred W; Taylor, Robert W; Prokisch, Holger; Kim, Jung-Ja P; Coon, Joshua J; Pagliarini, David J

    2016-08-18

    Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function. PMID:27499296

  6. ProtPhylo: identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling.

    PubMed

    Cheng, Yiming; Perocchi, Fabiana

    2015-07-01

    ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org.

  7. Optimizing an emperical scoring function for transmembrane protein structure determination.

    SciTech Connect

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  8. Deducing protein function by forensic integrative cell biology.

    PubMed

    Earnshaw, William C

    2013-12-01

    Our ability to sequence genomes has provided us with near-complete lists of the proteins that compose cells, tissues, and organisms, but this is only the beginning of the process to discover the functions of cellular components. In the future, it's going to be crucial to develop computational analyses that can predict the biological functions of uncharacterised proteins. At the same time, we must not forget those fundamental experimental skills needed to confirm the predictions or send the analysts back to the drawing board to devise new ones.

  9. Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression

    SciTech Connect

    Yoshikawa, Noritada; Shimizu, Noriaki; Sano, Motoaki; Ohnuma, Kei; Iwata, Satoshi; Hosono, Osamu; Fukuda, Keiichi; Morimoto, Chikao

    2008-06-20

    We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor {gamma}-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses.

  10. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  11. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean

    PubMed Central

    Zhou, Yuan; Yang, Yan; Zhou, Xinjian; Chi, Yingjun; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution. PMID:27708406

  12. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  13. PROFESS: a PROtein function, evolution, structure and sequence database.

    PubMed

    Triplet, Thomas; Shortridge, Matthew D; Griep, Mark A; Stark, Jaime L; Powers, Robert; Revesz, Peter

    2010-07-06

    The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are approximately 1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein-protein interaction networks. Database URL: http://cse.unl.edu/~profess/

  14. Design of Light-Controlled Protein Conformations and Functions.

    PubMed

    Ritterson, Ryan S; Hoersch, Daniel; Barlow, Kyle A; Kortemme, Tanja

    2016-01-01

    In recent years, interest in controlling protein function with light has increased. Light offers a number of unique advantages over other methods, including spatial and temporal control and high selectivity. Here, we describe a general protocol for engineering a protein to be controllable with light via reaction with an exogenously introduced photoisomerizable small molecule and illustrate our protocol with two examples from the literature: the engineering of the calcium affinity of the cell-cell adhesion protein cadherin, which is an example of a protein that switches from a native to a disrupted state (Ritterson et al. J Am Chem Soc (2013) 135:12516-12519), and the engineering of the opening and closing of the chaperonin Mm-cpn, an example of a switch between two functional states (Hoersch et al.: Nat Nanotechn (2013) 8:928-932). This protocol guides the user from considering which proteins may be most amenable to this type of engineering, to considerations of how and where to make the desired changes, to the assays required to test for functionality. PMID:27094293

  15. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT. PMID:22118932

  16. Protein engineering of Cas9 for enhanced function

    PubMed Central

    Oakes, Benjamin L.; Nadler, Dana C.; Savage, David F.

    2015-01-01

    CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complimentary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted directly into the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome modifying tools. PMID:25398355

  17. Evolutionary Insights into Premetazoan Functions of the Neuronal Protein Homer

    PubMed Central

    Burkhardt, Pawel; Grønborg, Mads; McDonald, Kent; Sulur, Tara; Wang, Qi; King, Nicole

    2014-01-01

    Reconstructing the evolution and ancestral functions of synaptic proteins promises to shed light on how neurons first evolved. The postsynaptic density (PSD) protein Homer scaffolds membrane receptors and regulates Ca2+ signaling in diverse metazoan cell types (including neurons and muscle cells), yet its ancestry and core functions are poorly understood. We find that the protein domain organization and essential biochemical properties of metazoan Homer proteins, including their ability to tetramerize, are conserved in the choanoflagellate Salpingoeca rosetta, one of the closest living relatives of metazoans. Unlike in neurons, Homer localizes to the nucleoplasm in S. rosetta and interacts directly with Flotillin, a protein more commonly associated with cell membranes. Surprisingly, we found that the Homer/Flotillin interaction and its localization to the nucleus are conserved in metazoan astrocytes. These findings suggest that Homer originally interacted with Flotillin in the nucleus of the last common ancestor of metazoans and choanoflagellates and was later co-opted to function as a membrane receptor scaffold in the PSD. PMID:24899667

  18. Functional and Structural Analysis of the Conserved EFhd2 Protein

    PubMed Central

    Acosta, Yancy Ferrer; Rodríguez Cruz, Eva N.; Vaquer, Ana del C.; Vega, Irving E.

    2013-01-01

    EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize the molecular requirements for EFhd2’s calcium binding activity. The results showed that mutations of a conserved aspartate on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity. Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this novel protein is thermostable. EFhd2’s thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium binding restored EFhd2’s thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins. PMID:22973849

  19. Identification of giant Mimivirus protein functions using RNA interference

    PubMed Central

    Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

    2015-01-01

    Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases. PMID:25972846

  20. From protein structure to function via single crystal optical spectroscopy

    PubMed Central

    Ronda, Luca; Bruno, Stefano; Bettati, Stefano; Storici, Paola; Mozzarelli, Andrea

    2015-01-01

    The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts,” including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms. PMID:25988179

  1. PSCL: predicting protein subcellular localization based on optimal functional domains.

    PubMed

    Wang, Kai; Hu, Le-Le; Shi, Xiao-He; Dong, Ying-Song; Li, Hai-Peng; Wen, Tie-Qiao

    2012-01-01

    It is well known that protein subcellular localizations are closely related to their functions. Although many computational methods and tools are available from Internet, it is still necessary to develop new algorithms in this filed to gain a better understanding of the complex mechanism of plant subcellular localization. Here, we provide a new web server named PSCL for plant protein subcellular localization prediction by employing optimized functional domains. After feature optimization, 848 optimal functional domains from InterPro were obtained to represent each protein. By calculating the distances to each of the seven categories, PSCL showing the possibilities of a protein located into each of those categories in ascending order. Toward our dataset, PSCL achieved a first-order predicted accuracy of 75.7% by jackknife test. Gene Ontology enrichment analysis showing that catalytic activity, cellular process and metabolic process are strongly correlated with the localization of plant proteins. Finally, PSCL, a Linux Operate System based web interface for the predictor was designed and is accessible for public use at http://pscl.biosino.org/.

  2. Protein requirements: from nitrogen balance to functional impact.

    PubMed

    Reeds, P J; Hutchens, T W

    1994-09-01

    Today because of tools capable of probing the details of metabolism, we are able to study the utilization of amino acids in specific, physiologically important processes. Such information may lead to recommended dietary allowances for each amino acid specific for subpopulations and perhaps individuals. These new findings identify the metabolic end-products of some amino acids as crucial intermediates in the maintenance of a variety of physiological functions that bear no direct relationship to protein metabolism. The precursor amino acids for these intermediate compounds are either non-essential or conditionally essential and thus may play a specific, non-protein, role in amino acid requirements in individuals close to nitrogen equilibrium. Another exciting area arises from the marked dissimilarity between the amino acid composition of milk proteins and other body proteins, suggesting that some milk proteins may have evolved to serve unique functions through specific digestion products. Such products may be important in micronutrient absorption in the neonate. Thus, amino acid requirements appear to reach beyond the tradition areas of nitrogen and protein metabolism. PMID:8089745

  3. The Cockayne syndrome group B protein is a functional dimer.

    PubMed

    Christiansen, Mette; Thorslund, Tina; Jochimsen, Bjarne; Bohr, Vilhelm A; Stevnsner, Tinna

    2005-09-01

    Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by developmental abnormalities, UV sensitivity, and premature aging. The CS group B (CSB) protein belongs to the SNF2-family of DNA-dependent ATPases and is implicated in transcription elongation, transcription coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. We demonstrate for the first time that full-length CSB positively cooperates in ATP hydrolysis as a function of protein concentration. We have investigated the quaternary structure of CSB using a combination of protein-protein complex trapping experiments and gel filtration, and found that CSB forms a dimer in solution. Chromatography studies revealed that enzymatically active CSB has an apparent molecular mass of approximately 360 kDa, consistent with dimerization of CSB. Importantly, in vivo protein cross-linking showed the presence of the CSB dimer in the nucleus of HeLa cells. We further show that dimerization occurs through the central ATPase domain of the protein. These results have implications for the mechanism of action of CSB, and suggest that other SNF2-family members might also function as dimers. PMID:16128801

  4. Insights into prion protein function from atomistic simulations.

    PubMed

    Hodak, Miroslav; Bernholc, Jerzy

    2010-01-01

    Computer simulations are a powerful tool for studies of biological systems. They have often been used to study prion protein (PrP), a protein responsible for neurodegenerative diseases, which include "mad cow disease" in cattle and Creutzfeldt-Jacob disease in humans. An important aspect of the prion protein is its interaction with copper ion, which is thought to be relevant for PrP's yet undetermined function and also potentially play a role in prion diseases. for studies of copper attachment to the prion protein, computer simulations have often been used to complement experimental data and to obtain binding structures of Cu-PrP complexes. This paper summarizes the results of recent ab initio calculations of copper-prion protein interactions focusing on the recently discovered concentration-dependent binding modes in the octarepeat region of this protein. In addition to determining the binding structures, computer simulations were also used to make predictions about PrP's function and the role of copper in prion diseases. The results demonstrate the predictive power and applicability of ab initio simulations for studies of metal-biomolecular complexes. PMID:20118658

  5. Regulation of thrombosis and vascular function by protein methionine oxidation.

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

  6. Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation

    PubMed Central

    Gsponer, Jörg; Babu, M. Madan

    2012-01-01

    Summary Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

  7. Insights into prion protein function from atomistic simulations

    PubMed Central

    Hodak, Miroslav

    2010-01-01

    Computer simulations are a powerful tool for studies of biological systems. They have often been used to study prion protein (PrP), a protein responsible for neurodegenerative diseases, which include “mad cow disease” in cattle and Creutzfeldt-Jacob disease in humans. An important aspect of the prion protein is its interaction with copper ion, which is thought to be relevant for PrP’s yet undetermined function and also potentially play a role in prion diseases. For studies of copper attachment to the prion protein, computer simulations have often been used to complement experimental data and to obtain binding structures of Cu-PrP complexes. This paper summarizes the results of recent ab initio calculations of copper-prion protein interactions focusing on the recently discovered concentration-dependent binding modes in the octarepeat region of this protein. In addition to determining the binding structures, computer simulations were also used to make predictions about PrP’s function and the role of copper in prion diseases. The results demonstrate the predictive power and applicability of ab initio simulations for studies of metal-biomolecular complexes. PMID:20118658

  8. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

    PubMed

    Wu, Nicholas C; Olson, C Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-07-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.

  9. Structure and Function of Transient Encounters of Redox Proteins.

    PubMed

    Volkov, Alexander N

    2015-12-15

    Many biomolecular interactions proceed via lowly populated, transient intermediates. Believed to facilitate formation of a productive complex, these short-lived species are inaccessible to conventional biophysical and structural techniques and, until recently, could only be studied by theoretical simulations. Recent development of experimental approaches sensitive to the presence of minor species--in particular paramagnetic relaxation enhancement (PRE) NMR spectroscopy--has enabled direct visualization and detailed characterization of such lowly populated states. Collectively referred to as an encounter complex, the binding intermediates are particularly important in transient protein interactions, such as those orchestrating signaling cascades or energy-generating electron transfer (ET) chains. Here I discuss encounter complexes of redox proteins mediating biological ET reactions, which are essential for many vital cellular activities including oxidative phosphorylation and photosynthesis. In particular, this Account focuses on the complex of cytochrome c (Cc) and cytochrome c peroxidase (CcP), which is a paradigm of biomolecular ET and an attractive system for studying protein binding and enzymatic catalysis. The Cc-CcP complex formation proceeds via an encounter state, consisting of multiple protein-protein orientations sampled in the search of the dominant, functionally active bound form and exhibiting a broad spatial distribution, in striking agreement with earlier theoretical simulations. At low ionic strength, CcP binds another Cc molecule to form a weak ternary complex, initially inferred from kinetics experiments and postulated to account for the measured ET activity. Despite strenuous efforts, the ternary complex could not be observed directly and remained eagerly sought for the past two decades. Very recently, we have solved its structure in solution and shown that it consists of two binding forms: the dominant, ET-inactive geometry and an ensemble of

  10. Structure and Function of Transient Encounters of Redox Proteins.

    PubMed

    Volkov, Alexander N

    2015-12-15

    Many biomolecular interactions proceed via lowly populated, transient intermediates. Believed to facilitate formation of a productive complex, these short-lived species are inaccessible to conventional biophysical and structural techniques and, until recently, could only be studied by theoretical simulations. Recent development of experimental approaches sensitive to the presence of minor species--in particular paramagnetic relaxation enhancement (PRE) NMR spectroscopy--has enabled direct visualization and detailed characterization of such lowly populated states. Collectively referred to as an encounter complex, the binding intermediates are particularly important in transient protein interactions, such as those orchestrating signaling cascades or energy-generating electron transfer (ET) chains. Here I discuss encounter complexes of redox proteins mediating biological ET reactions, which are essential for many vital cellular activities including oxidative phosphorylation and photosynthesis. In particular, this Account focuses on the complex of cytochrome c (Cc) and cytochrome c peroxidase (CcP), which is a paradigm of biomolecular ET and an attractive system for studying protein binding and enzymatic catalysis. The Cc-CcP complex formation proceeds via an encounter state, consisting of multiple protein-protein orientations sampled in the search of the dominant, functionally active bound form and exhibiting a broad spatial distribution, in striking agreement with earlier theoretical simulations. At low ionic strength, CcP binds another Cc molecule to form a weak ternary complex, initially inferred from kinetics experiments and postulated to account for the measured ET activity. Despite strenuous efforts, the ternary complex could not be observed directly and remained eagerly sought for the past two decades. Very recently, we have solved its structure in solution and shown that it consists of two binding forms: the dominant, ET-inactive geometry and an ensemble of

  11. Function of prokaryotic and eukaryotic ABC proteins in lipid transport.

    PubMed

    Pohl, Antje; Devaux, Philippe F; Herrmann, Andreas

    2005-03-21

    ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry. PMID:15749056

  12. Modification of protein structure and function using photoactivated porphyrin ligands

    NASA Astrophysics Data System (ADS)

    Moreno, Gabriel

    2015-03-01

    The tremendous advances in genomic research have sparked an interest in investigating the possibility to ``manipulate'' the structure of proteins that modify existing functionality. This study makes use of small molecules (e.g., porphyrins) to photosensitize proteins and modify the higher order structure of the polypeptide with the goal of engineering novel functions, or affecting/eliminating native functions. The irradiation of non-covalently bound ligands prompts charge transfer events that have the potential to locally modify the structure of the host protein. The characterization of photoinduced conformational changes in the protein/porphyrin complex is carried out using a combination of electronic spectroscopy and kinetics (e.g., fluorescence spectroscopy, fluorescence decay, circular dichroism). This study is focused primarily on human serum albumin (HSA) as a model. The structure of HSA is well established, the binding sites for an array of ligands are well characterized (including one for protoporphyrins), and HSA provides a series of functions (including some allosteric activity) that can be tested.

  13. Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein-Protein Interaction Data.

    PubMed

    Pokharel, Yuba Raj; Saarela, Jani; Szwajda, Agnieszka; Rupp, Christian; Rokka, Anne; Lal Kumar Karna, Shibendra; Teittinen, Kaisa; Corthals, Garry; Kallioniemi, Olli; Wennerberg, Krister; Aittokallio, Tero; Westermarck, Jukka

    2015-12-01

    High content protein interaction screens have revolutionized our understanding of protein complex assembly. However, one of the major challenges in translation of high content protein interaction data is identification of those interactions that are functionally relevant for a particular biological question. To address this challenge, we developed a relevance ranking platform (RRP), which consist of modular functional and bioinformatic filters to provide relevance rank among the interactome proteins. We demonstrate the versatility of RRP to enable a systematic prioritization of the most relevant interaction partners from high content data, highlighted by the analysis of cancer relevant protein interactions for oncoproteins Pin1 and PME-1. We validated the importance of selected interactions by demonstration of PTOV1 and CSKN2B as novel regulators of Pin1 target c-Jun phosphorylation and reveal previously unknown interacting proteins that may mediate PME-1 effects via PP2A-inhibition. The RRP framework is modular and can be modified to answer versatile research problems depending on the nature of the biological question under study. Based on comparison of RRP to other existing filtering tools, the presented data indicate that RRP offers added value especially for the analysis of interacting proteins for which there is no sufficient prior knowledge available. Finally, we encourage the use of RRP in combination with either SAINT or CRAPome computational tools for selecting the candidate interactors that fulfill the both important requirements, functional relevance, and high confidence interaction detection.

  14. PPR-SMRs: ancient proteins with enigmatic functions.

    PubMed

    Liu, Sheng; Melonek, Joanna; Boykin, Laura M; Small, Ian; Howell, Katharine A

    2013-01-01

    A small subset of the large pentatricopeptide repeat (PPR) protein family in higher plants contain a C-terminal small MutS-related (SMR) domain. Although few in number, they figure prominently in the chloroplast biogenesis and retrograde signaling literature due to their striking mutant phenotypes. In this review, we summarize current knowledge of PPR-SMR proteins focusing on Arabidopsis and maize proteomic and mutant studies. We also examine their occurrence in other organisms and have determined by phylogenetic analysis that, while they are limited to species that contain chloroplasts, their presence in algae and early branching land plant lineages indicates that the coupling of PPR motifs and an SMR domain into a single protein occurred early in the evolution of the Viridiplantae clade. In addition, we discuss their possible function and have examined conservation between SMR domains from Arabidopsis PPR proteins with those from other species that have been shown to possess endonucleolytic activity.

  15. Fundamental Characteristics of AAA+ Protein Family Structure and Function

    PubMed Central

    2016-01-01

    Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins. PMID:27703410

  16. Structure and function of seed lipid-body-associated proteins.

    PubMed

    Purkrtova, Zita; Jolivet, Pascale; Miquel, Martine; Chardot, Thierry

    2008-10-01

    Many organisms among the different kingdoms store reserve lipids in discrete subcellular organelles called lipid bodies. In plants, lipid bodies can be found in seeds but also in fruits (olives, ...), and in leaves (plastoglobules). These organelles protect plant lipid reserves against oxidation and hydrolysis until seed germination and seedling establishment. They can be stabilized by specific structural proteins, namely the oleosins and caleosins, which act as natural emulsifiers. Considering the putative role of some of them in controlling the size of lipid bodies, these proteins may constitute important targets for seed improvement both in term of oil seed yield and optimization of technological processes for extraction of oil and storage proteins. We present here an overview of the data on the structure of these proteins, which are scarce, and sometimes contradictory and on their functional roles. PMID:18926488

  17. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  18. Protein complexes and functional modules in molecular networks

    NASA Astrophysics Data System (ADS)

    Spirin, Victor; Mirny, Leonid A.

    2003-10-01

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  19. A surprising role for conformational entropy in protein function

    PubMed Central

    Wand, A. Joshua; Moorman, Veronica R.; Harpole, Kyle W.

    2014-01-01

    Formation of high-affinity complexes is critical for the majority of enzymatic reactions involving proteins. The creation of the family of Michaelis and other intermediate complexes during catalysis clearly involves a complicated manifold of interactions that are diverse and complex. Indeed, computing the energetics of interactions between proteins and small molecule ligands using molecular structure alone remains a grand challenge. One of the most difficult contributions to the free energy of protein-ligand complexes to experimentally access is that due to changes in protein conformational entropy. Fortunately, recent advances in solution nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. This review briefly summarizes the experimental approaches currently employed to characterize fast internal motion in proteins, how this information is used to gain insight into conformational entropy, what has been learned and what the future may hold for this emerging view of protein function. PMID:23478875

  20. cncRNAs: Bi-functional RNAs with protein coding and non-coding functions

    PubMed Central

    Kumari, Pooja; Sampath, Karuna

    2015-01-01

    For many decades, the major function of mRNA was thought to be to provide protein-coding information embedded in the genome. The advent of high-throughput sequencing has led to the discovery of pervasive transcription of eukaryotic genomes and opened the world of RNA-mediated gene regulation. Many regulatory RNAs have been found to be incapable of protein coding and are hence termed as non-coding RNAs (ncRNAs). However, studies in recent years have shown that several previously annotated non-coding RNAs have the potential to encode proteins, and conversely, some coding RNAs have regulatory functions independent of the protein they encode. Such bi-functional RNAs, with both protein coding and non-coding functions, which we term as ‘cncRNAs’, have emerged as new players in cellular systems. Here, we describe the functions of some cncRNAs identified from bacteria to humans. Because the functions of many RNAs across genomes remains unclear, we propose that RNAs be classified as coding, non-coding or both only after careful analysis of their functions. PMID:26498036

  1. Desensitization of G protein-coupled receptors and neuronal functions.

    PubMed

    Gainetdinov, Raul R; Premont, Richard T; Bohn, Laura M; Lefkowitz, Robert J; Caron, Marc G

    2004-01-01

    G protein-coupled receptors (GPCRs) have proven to be the most highly favorable class of drug targets in modern pharmacology. Over 90% of nonsensory GPCRs are expressed in the brain, where they play important roles in numerous neuronal functions. GPCRs can be desensitized following activation by agonists by becoming phosphorylated by members of the family of G protein-coupled receptor kinases (GRKs). Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways. Discussed in this review are recent progress in understanding basics of GPCR desensitization, novel functional roles, patterns of brain expression, and receptor specificity of GRKs and beta arrestins in major brain functions. In particular, screening of genetically modified mice lacking individual GRKs or beta arrestins for alterations in behavioral and biochemical responses to cocaine and morphine has revealed a functional specificity in dopamine and mu-opioid receptor regulation of locomotion and analgesia. An important and specific role of GRKs and beta arrestins in regulating physiological responsiveness to psychostimulants and morphine suggests potential involvement of these molecules in certain brain disorders, such as addiction, Parkinson's disease, mood disorders, and schizophrenia. Furthermore, the utility of a pharmacological strategy aimed at targeting this GPCR desensitization machinery to regulate brain functions can be envisaged. PMID:15217328

  2. Biomolecular templating of functional hybrid nanostructures using repeat protein scaffolds.

    PubMed

    Romera, David; Couleaud, Pierre; Mejias, Sara H; Aires, Antonio; Cortajarena, Aitziber L

    2015-10-01

    The precise synthesis of materials and devices with tailored complex structures and properties is a requisite for the development of the next generation of products based on nanotechnology. Nowadays, the technology for the generation of this type of devices lacks the precision to determine their properties and is accomplished mostly by 'trial and error' experimental approaches. The use of bottom-up approaches that rely on highly specific biomolecular interactions of small and simple components is an attractive approach for the templating of nanoscale elements. In nature, protein assemblies define complex structures and functions. Engineering novel bio-inspired assemblies by exploiting the same rules and interactions that encode the natural diversity is an emerging field that opens the door to create nanostructures with numerous potential applications in synthetic biology and nanotechnology. Self-assembly of biological molecules into defined functional structures has a tremendous potential in nano-patterning and the design of novel materials and functional devices. Molecular self-assembly is a process by which complex 3D structures with specified functions are constructed from simple molecular building blocks. Here we discuss the basis of biomolecular templating, the great potential of repeat proteins as building blocks for biomolecular templating and nano-patterning. In particular, we focus on the designed consensus tetratricopeptide repeats (CTPRs), the control on the assembly of these proteins into higher order structures and their potential as building blocks in order to generate functional nanostructures and materials.

  3. Senescence Marker Protein 30: Functional and Structural Insights to its Unknown Physiological Function

    PubMed Central

    Scott, Stephanie H.; Bahnson, Brian J.

    2011-01-01

    Senescence marker protein 30 (SMP30) is a multifunctional protein involved in cellular Ca2+ homeostasis and the biosynthesis of ascorbate in non-primate mammals. The primary structure of the protein is highly conserved among vertebrates, suggesting the existence of a significant physiological function common to all mammals, including primates. Enzymatic activities of SMP30 include aldonolactone and organophosphate hydrolysis. Protective effects against apoptosis and oxidative stress have been reported. X-ray crystallography revealed that SMP30 is a six-bladed β-propeller with structural similarity to paraoxonase 1, another protein with lactonase and organophosphate hydrolase activities. SMP30 has recently been tied to several physiological conditions including osteoporosis, liver fibrosis, diabetes, and cancer. This review aims to describe the recent advances made toward understanding the connection between molecular structure, enzymatic activity and physiological function of this highly conserved, multifaceted protein. PMID:22844387

  4. Expressed Protein Ligation: A Resourceful Tool to Study Protein Structure and Function

    PubMed Central

    Berrade, Luis; Camarero, Julio A.

    2013-01-01

    This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function. PMID:19685006

  5. Bioengineered silk proteins to control cell and tissue functions.

    PubMed

    Preda, Rucsanda C; Leisk, Gary; Omenetto, Fiorenzo; Kaplan, David L

    2013-01-01

    Silks are defined as protein polymers that are spun into fibers by some lepidoptera larvae such as silkworms, spiders, scorpions, mites, and flies. Silk proteins are usually produced within specialized glands in these animals after biosynthesis in epithelial cells that line the glands, followed by secretion into the lumen of the gland prior to spinning into fibers.The most comprehensively characterized silks are from the domesticated silkworm (Bombyx mori) and from some spiders (Nephila clavipes and Araneus diadematus). Silkworm silk has been used commercially as biomedical sutures for decades and in textile production for centuries. Because of their impressive mechanical properties, silk proteins provide an important set of material options in the fields of controlled drug release, and for biomaterials and scaffolds for tissue engineering. Silkworm silk from B. mori consists primarily of two protein components, fibroin, the structural protein of silk fibers, and sericins, the water-soluble glue-like proteins that bind the fibroin fibers together. Silk fibroin consists of heavy and light chain polypeptides linked by a disulfide bond. Fibroin is the protein of interest for biomedical materials and it has to be purified/extracted from the silkworm cocoon by removal of the sericin. Characteristics of silks, including biodegradability, biocompatibility, controllable degradation rates, and versatility to generate different material formats from gels to fibers and sponges, have attracted interest in the field of biomaterials. Cell culture and tissue formation using silk-based biomaterials have been pursued, where appropriate cell adhesion, proliferation, and differentiation on or in silk biomaterials support the regeneration of tissues. The relative ease with which silk proteins can be processed into a variety of material morphologies, versatile chemical functionalization options, processing in water or solvent, and the related biological features of biocompatibility and

  6. Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

    PubMed Central

    VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

    2011-01-01

    Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

  7. Computational design of receptor and sensor proteins with novel functions

    NASA Astrophysics Data System (ADS)

    Looger, Loren L.; Dwyer, Mary A.; Smith, James J.; Hellinga, Homme W.

    2003-05-01

    The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, L-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or L-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.

  8. The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks*

    PubMed Central

    Van Itallie, Christina M.; Aponte, Angel; Tietgens, Amber Jean; Gucek, Marjan; Fredriksson, Karin; Anderson, James Melvin

    2013-01-01

    The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization. PMID:23553632

  9. Different Electrostatic Potentials Define ETGE and DLG Motifs as Hinge and Latch in Oxidative Stress Response▿

    PubMed Central

    Tong, Kit I.; Padmanabhan, Balasundaram; Kobayashi, Akira; Shang, Chengwei; Hirotsu, Yosuke; Yokoyama, Shigeyuki; Yamamoto, Masayuki

    2007-01-01

    Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress. PMID:17785452

  10. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry.

    PubMed

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application. PMID:26761849

  11. Enzymatic functionalization of a nanobody using protein insertion technology.

    PubMed

    Crasson, O; Rhazi, N; Jacquin, O; Freichels, A; Jérôme, C; Ruth, N; Galleni, M; Filée, P; Vandevenne, M

    2015-10-01

    Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays. PMID:25852149

  12. Gene evolution and functions of extracellular matrix proteins in teeth

    PubMed Central

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-01-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  13. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry.

    PubMed

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application.

  14. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry

    PubMed Central

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application. PMID:26761849

  15. Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila

    PubMed Central

    Gaertig, Jacek; Wloga, Dorota; Vasudevan, Krishna Kumar; Guha, Mayukh; Dentler, William

    2015-01-01

    The ciliate Tetrahymena thermophila is an excellent model system for the discovery and functional studies of ciliary proteins. The power of the model is based on the ease with which cilia can be purified in large quantities for fractionation and proteomic identification, and the ability to knock out any gene by homologous DNA recombination. Here, we include methods used by our laboratories for isolation and fractionation of cilia, in vivo tagging and localization of ciliary proteins and the evaluation of ciliary mutants. PMID:23522474

  16. Functional roles of HIV-1 Tat protein in the nucleus.

    PubMed

    Musinova, Yana R; Sheval, Eugene V; Dib, Carla; Germini, Diego; Vassetzky, Yegor S

    2016-02-01

    Human immunodeficiency virus-1 (HIV-1) Tat protein is one of the most important regulatory proteins for viral gene expression in the host cell and can modulate different cellular processes. In addition, Tat is secreted by the infected cell and can be internalized by neighboring cells; therefore, it affects both infected and uninfected cells. Tat can modulate cellular processes by interacting with different cellular structures and signaling pathways. In the nucleus, Tat might be localized either in the nucleoplasm or the nucleolus depending on its concentration. Here we review the distinct functions of Tat in the nucleoplasm and the nucleolus in connection with viral infection and HIV-induced oncogenesis. PMID:26507246

  17. Eliciting the Functional Taxonomy from protein annotations and taxa.

    PubMed

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-08-18

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules.

  18. Eliciting the Functional Taxonomy from protein annotations and taxa

    PubMed Central

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-01-01

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules. PMID:27534507

  19. Eliciting the Functional Taxonomy from protein annotations and taxa.

    PubMed

    Falda, Marco; Lavezzo, Enrico; Fontana, Paolo; Bianco, Luca; Berselli, Michele; Formentin, Elide; Toppo, Stefano

    2016-01-01

    The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules. PMID:27534507

  20. Preparation, characterization and functional properties of flax seed protein isolate.

    PubMed

    Kaushik, Pratibha; Dowling, Kim; McKnight, Stafford; Barrow, Colin J; Wang, Bo; Adhikari, Benu

    2016-04-15

    Flaxseed protein isolate (FPI) was extracted from flaxseeds, and its amino acid composition and functional properties (solubility, thermal stability, emulsifying properties and electrostatic charge density, water holding and fat absorption capacities) were determined. The highest purity of FPI (90.6%) was achieved by extraction at 60°C. FPI had a low lysine to arginine ratio of 0.25, which is desired in heart-healthy foods and infant formulas. The denaturation temperature of FPI was 105°C. FPI had the highest emulsion activity index (375.51 m(2)/g), highest emulsion stability index (179.5 h) and zeta potential (-67.4 mV) when compared to those of other commonly used proteins, such as sodium caseinate (SC), whey protein isolate (WPI), gelatin (Gel) and soy protein isolate (SPI). The average emulsion droplet size of emulsions stabilized by these proteins was in the order SCproteins. PMID:26616943

  1. Preparation, characterization and functional properties of flax seed protein isolate.

    PubMed

    Kaushik, Pratibha; Dowling, Kim; McKnight, Stafford; Barrow, Colin J; Wang, Bo; Adhikari, Benu

    2016-04-15

    Flaxseed protein isolate (FPI) was extracted from flaxseeds, and its amino acid composition and functional properties (solubility, thermal stability, emulsifying properties and electrostatic charge density, water holding and fat absorption capacities) were determined. The highest purity of FPI (90.6%) was achieved by extraction at 60°C. FPI had a low lysine to arginine ratio of 0.25, which is desired in heart-healthy foods and infant formulas. The denaturation temperature of FPI was 105°C. FPI had the highest emulsion activity index (375.51 m(2)/g), highest emulsion stability index (179.5 h) and zeta potential (-67.4 mV) when compared to those of other commonly used proteins, such as sodium caseinate (SC), whey protein isolate (WPI), gelatin (Gel) and soy protein isolate (SPI). The average emulsion droplet size of emulsions stabilized by these proteins was in the order SCproteins.

  2. Prediction of functional sites in proteins using conserved functional group analysis.

    PubMed

    Innis, C Axel; Anand, A Prem; Sowdhamini, R

    2004-04-01

    A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects. PMID:15033369

  3. Structure-based inference of molecular functions of proteins of unknown function from Berkeley Structural Genomics Center

    SciTech Connect

    Kim, Sung-Hou; Shin, Dong Hae; Hou, Jingtong; Chandonia, John-Marc; Das, Debanu; Choi, In-Geol; Kim, Rosalind; Kim, Sung-Hou

    2007-09-02

    Advances in sequence genomics have resulted in an accumulation of a huge number of protein sequences derived from genome sequences. However, the functions of a large portion of them cannot be inferred based on the current methods of sequence homology detection to proteins of known functions. Three-dimensional structure can have an important impact in providing inference of molecular function (physical and chemical function) of a protein of unknown function. Structural genomics centers worldwide have been determining many 3-D structures of the proteins of unknown functions, and possible molecular functions of them have been inferred based on their structures. Combined with bioinformatics and enzymatic assay tools, the successful acceleration of the process of protein structure determination through high throughput pipelines enables the rapid functional annotation of a large fraction of hypothetical proteins. We present a brief summary of the process we used at the Berkeley Structural Genomics Center to infer molecular functions of proteins of unknown function.

  4. Prebiotic Alternatives to Proteins: Structure and Function of Hyperbranched Polyesters

    NASA Astrophysics Data System (ADS)

    Mamajanov, Irena; Callahan, Michael P.; Dworkin, Jason P.; Cody, George D.

    2015-06-01

    Proteins are responsible multiple biological functions, such as ligand binding, catalysis, and ion channeling. This functionality is enabled by proteins' three-dimensional structures that require long polypeptides. Since plausibly prebiotic synthesis of functional polypeptides has proven challenging in the laboratory, we propose that these functions may have been initially performed by alternative macromolecular constructs, namely hyperbranched polymers (HBPs), during early stages of chemical evolution. HBPs can be straightforwardly synthesized in one-pot processes, possess globular structures determined by their architecture as opposed to folding in proteins, and have documented ligand binding and catalytic properties. Our initial study focuses on glycerol-citric acid HBPs synthesized via moderate heating in the dry state. The polymerization products consisted of a mixture of isomeric structures of varying molar mass as evidenced by NMR, mass spectrometry and size-exclusion chromatography. Addition of divalent cations during polymerization resulted in increased incorporation of citric acid into the HBPs and the possible formation of cation-oligomer complexes. The chelating properties of citric acid govern the makeup of the resulting polymer, turning the polymerization system into a rudimentary smart material.

  5. C1 inhibitor hinge region mutations produce dysfunction by different mechanisms.

    PubMed

    Davis, A E; Aulak, K; Parad, R B; Stecklein, H P; Eldering, E; Hack, C E; Kramer, J; Strunk, R C; Bissler, J; Rosen, F S

    1992-08-01

    Heterozygosity for a mutant dysfunctional C1 inhibitor protein, a member of the serine proteinase inhibitor (serpin) superfamily, results in type II hereditary angioneurotic oedema. We identified a "hinge" region mutation in C1 inhibitor with a Val to Glu replacement at P14 Val-432. Recombinant C1 inhibitors P10 Ala-->Thr and P14Val-->Glu did not form stable complexes with fluid phase C1s or kallikrein. The P14 Val-->Glu mutant, however, was cleaved to a 96K form by C1s, while the P10 Ala-->Thr mutant was not. The recombinant P10 mutant also did not complex with C1s, kallikrein or beta-factor Xlla-Sepharose. The two mutations, therefore, result in dysfunction by different mechanisms: in one (P14 Val-->Glu), the inhibitor is converted to a substrate, while in the other (P10 Ala-->Thr), interaction with target protease is blocked.

  6. The hinge region fragment of immunoglobulin G improves immunogenicity of recombinant gonadotrophin-releasing hormone conjugated to the T-helper epitope in designing peptide vaccines

    PubMed Central

    Xu, Jinshu; Wu, Jie; Wang, Xuejun; Zhang, Yin; Li, Wenjia; Zhu, Zheng; Zhu, Dongya; Hu, Zhuoyi; Roque, Rouel S; Liu, Jingjing

    2009-01-01

    In our previous study, the hinge fragment (225–232/225′–232′) of human immunoglobulin G1 (IgG1) was used as a space peptide linker for synthesizing the GnRH3–hinge–MVP chimeric peptide, whereby three repeated gonadotrophin-releasing hormone (GnRH) units and a T-cell epitope from measles virus fusion protein (MVP) were amide-bond-linked at the N and C terminus, respectively, to the hinge peptide for producing anti-GnRH antibody responses. To investigate whether or not the hinge region fragment can improve the immunogenicity of GnRH, we further synthesized and purified GnRH3–hinge–MVP, GnRH3–hinge and GnRH3–MVP using recombinant DNA technology. Under high pH conditions, GnRH3–hinge–MVP was capable of forming double-chain structures. Immunization of male mice with the immunogens of GnRH3–hinge–MVP resulted in the generation of high-titre antibodies specific for GnRH. The synthetic GnRH3–hinge and GnRH3–MVP induced a lower titre of anti-GnRH antibody than GnRH3–hinge–MVP. This was followed by a decrease in serum testosterone levels, which resulted in a low level of expression of the relaxin-like factor gene in the testis. Our data suggest that peptide and T-cell epitopes oriented at the N-terminus or C-terminus of hinge peptides simplify the antigenic peptide conjugates and may be considered as potential synthetic immunogens. PMID:19740311

  7. Protein function in precision medicine: deep understanding with machine learning.

    PubMed

    Rost, Burkhard; Radivojac, Predrag; Bromberg, Yana

    2016-08-01

    Precision medicine and personalized health efforts propose leveraging complex molecular, medical and family history, along with other types of personal data toward better life. We argue that this ambitious objective will require advanced and specialized machine learning solutions. Simply skimming some low-hanging results off the data wealth might have limited potential. Instead, we need to better understand all parts of the system to define medically relevant causes and effects: how do particular sequence variants affect particular proteins and pathways? How do these effects, in turn, cause the health or disease-related phenotype? Toward this end, deeper understanding will not simply diffuse from deeper machine learning, but from more explicit focus on understanding protein function, context-specific protein interaction networks, and impact of variation on both. PMID:27423136

  8. Structure and Function of CW Domain Containing Proteins.

    PubMed

    Liu, Yanli; Liu, Shasha; Zhang, Xinxin; Liang, Xiao; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Deng, Lingfu; Yang, Jihong; Qi, Chao

    2016-01-01

    The CW domain is a zinc binding domain, composed of approximately 50- 60 amino acid residues with four conserved cysteine (C) and two to four conserved tryptophan (W) residues. The members of the superfamily of CW domain containing proteins, comprised of 12 different eukaryotic nuclear protein families, are extensively expressed in vertebrates, vertebrate-infecting parasites and higher plants, where they are often involved in chromatin remodeling, methylation recognition, epigenetic regulation and early embryonic development. Since the first CW domain structure was determined 5 years ago, structures of five CW domains have been solved so far. In this review, we will discuss these recent advances in understanding the identification, definition, structure, and functions of the CW domain containing proteins. PMID:26806410

  9. Multipolar functions of BCL-2 proteins link energetics to apoptosis

    PubMed Central

    Hardwick, J. Marie; Chen, Ying-bei; Jonas, Elizabeth A.

    2012-01-01

    Classical apoptotic cell death is now sufficiently well understood to be interrogated with mathematical modeling and to be skillfully manipulated with targeted drugs for clinical benefit. However, a biological black hole has emerged with the realization that apoptosis regulators are functionally multipolar. BCL-2 family proteins appear to have much greater effects on cells than can be explained by their known roles in apoptosis. While these effects may be observable simply because the cell is not dead, the general assumption is that BCL-2 proteins have yet undiscovered biochemical activities. Conversely, these yet uncharacterized day-jobs may underlie their profound effects on cell survival, challenging current assumptions about classical apoptosis. Even their sub-mitochondrial localizations remain controversial. Here we attempt to integrate seemingly conflicting information with the prospect that BCL-2 proteins themselves may be the critical crosstalk between life and death. PMID:22560661

  10. The Evolution of Function in Strictosidine Synthase-like Proteins

    PubMed Central

    Hicks, Michael A.; Barber, Alan E.; Giddings, Lesley-Ann; Caldwell, Jenna; O’Connor, Sarah E.; Babbitt, Patricia C.

    2013-01-01

    The exponential growth of sequence data provides abundant information for the discovery of new enzyme reactions. Correctly annotating the functions of highly diverse proteins can be difficult, however, hindering use of this information. Global analysis of large superfamilies of related proteins is a powerful strategy for understanding the evolution of reactions by identifying catalytic commonalities and differences in reaction and substrate specificity, even when only a few members have been biochemically or structurally characterized. A comparison of >2500 sequences sharing the six-bladed β-propeller fold establishes sequence, structural and functional links among the three subgroups of the functionally diverse N6P superfamily: the arylesterase-like and senescence marker protein-30/gluconolactonase/luciferin-regenerating enzyme-like (SGL) subgroups, representing enzymes that catalyze lactonase and related hydrolytic reactions, and the so-called “strictosidine synthase-like” (SSL) subgroup. Metal-coordinating residues were identified as broadly conserved in the active sites of all three subgroups except for a few proteins from the SSL subgroup, which have been experimentally determined to catalyze the quite different strictosidine synthase (SS) reaction, a metal-independent condensation reaction. Despite these differences, comparison of conserved catalytic features of the arylesterase-like and SGL enzymes with the SSs identified similar structural and mechanistic attributes between the hydrolytic reactions catalyzed by the former and the condensation reaction catalyzed by SS. The results also suggest that despite their annotations, the great majority of these >500 SSL sequences do not catalyze the SS reaction; rather, they likely catalyze hydrolytic reactions typical of the other two subgroups instead. This prediction was confirmed experimentally for one of these proteins. PMID:21948213

  11. Graphlet kernels for prediction of functional residues in protein structures.

    PubMed

    Vacic, Vladimir; Iakoucheva, Lilia M; Lonardi, Stefano; Radivojac, Predrag

    2010-01-01

    We introduce a novel graph-based kernel method for annotating functional residues in protein structures. A structure is first modeled as a protein contact graph, where nodes correspond to residues and edges connect spatially neighboring residues. Each vertex in the graph is then represented as a vector of counts of labeled non-isomorphic subgraphs (graphlets), centered on the vertex of interest. A similarity measure between two vertices is expressed as the inner product of their respective count vectors and is used in a supervised learning framework to classify protein residues. We evaluated our method on two function prediction problems: identification of catalytic residues in proteins, which is a well-studied problem suitable for benchmarking, and a much less explored problem of predicting phosphorylation sites in protein structures. The performance of the graphlet kernel approach was then compared against two alternative methods, a sequence-based predictor and our implementation of the FEATURE framework. On both tasks, the graphlet kernel performed favorably; however, the margin of difference was considerably higher on the problem of phosphorylation site prediction. While there is data that phosphorylation sites are preferentially positioned in intrinsically disordered regions, we provide evidence that for the sites that are located in structured regions, neither the surface accessibility alone nor the averaged measures calculated from the residue microenvironments utilized by FEATURE were sufficient to achieve high accuracy. The key benefit of the graphlet representation is its ability to capture neighborhood similarities in protein structures via enumerating the patterns of local connectivity in the corresponding labeled graphs.

  12. Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster

    PubMed Central

    Cassar, Marlène; Kretzschmar, Doris

    2016-01-01

    The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer’s Disease (AD) due to its cleavage resulting in the production of the Aβ peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including Drosophila, suggesting an important physiological function. Besides Aβ, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aβ, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation. PMID:27507933

  13. Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster.

    PubMed

    Cassar, Marlène; Kretzschmar, Doris

    2016-01-01

    The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer's Disease (AD) due to its cleavage resulting in the production of the Aβ peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including Drosophila, suggesting an important physiological function. Besides Aβ, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aβ, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation. PMID:27507933

  14. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    PubMed Central

    Millhauser, Glenn L.

    2010-01-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease. PMID:17076634

  15. Production of functional proteins: balance of shear stress and gravity

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2011-01-01

    A method for the production of functional proteins including hormones by renal cells in a three dimensional culturing process responsive to shear stress uses a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-.alpha.-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D.sub.3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating an in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  16. Production of functional proteins: balance of shear stress and gravity

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2007-01-01

    The present invention provides a method for production of functional proteins including hormones by renal cells in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-a-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  17. Production of functional proteins: balance of shear stress and gravity

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Kaysen, James Howard (Inventor)

    2004-01-01

    The present invention provides a method for production of functional proteins including hormones by renal cells in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-a-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

  18. Copper and the prion protein: methods, structures, function, and disease.

    PubMed

    Millhauser, Glenn L

    2007-01-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrP(C) to PrP(Sc). Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrP(C) in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrP(C), with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrP(C) function, and emerging connections between copper and prion disease. PMID:17076634

  19. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    NASA Astrophysics Data System (ADS)

    Millhauser, Glenn L.

    2007-05-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

  20. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    PubMed

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  1. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  2. Uncoupling proteins--a new family of proteins with unknown function.

    PubMed

    Erlanson-Albertsson, Charlotte

    2002-02-01

    Uncoupling proteins are inner mitochondrial membrane proteins, which dissipate the proton gradient, releasing the stored energy as heat. Five proteins have been cloned, named UCP1, UCP2, UCP3, UCP4 and UCP5/BMCP1. These proteins are structurally related but differ in tissue expression. UCP1 is expressed uniquely in the brown adipose tissue, while UCP2 is widely distributed, UCP3 is mainly restricted to skeletal muscle and UCP4 and UCP5/BMCP1 expressed in the brain. The properties and regulation of the uncoupling proteins and their exact function has been the focus of an intense research during recent years. This review briefly summarizes the actual knowledge of the properties and function of this new family of proteins. While UCP1 has a clear role in energy homeostasis, the newcomers UCP2-UCP5 may have more delicate physiological importance acting as free radical oxygen scavengers and in the regulation of ATP-dependent processes, such as secretion.

  3. Nuclear pore proteins and the control of genome functions

    PubMed Central

    Ibarra, Arkaitz

    2015-01-01

    Nuclear pore complexes (NPCs) are composed of several copies of ∼30 different proteins called nucleoporins (Nups). NPCs penetrate the nuclear envelope (NE) and regulate the nucleocytoplasmic trafficking of macromolecules. Beyond this vital role, NPC components influence genome functions in a transport-independent manner. Nups play an evolutionarily conserved role in gene expression regulation that, in metazoans, extends into the nuclear interior. Additionally, in proliferative cells, Nups play a crucial role in genome integrity maintenance and mitotic progression. Here we discuss genome-related functions of Nups and their impact on essential DNA metabolism processes such as transcription, chromosome duplication, and segregation. PMID:25691464

  4. Flow over a cylinder with a hinged-splitter plate

    NASA Astrophysics Data System (ADS)

    Shukla, S.; Govardhan, R. N.; Arakeri, J. H.

    2009-05-01

    Previous work on rigid splitter plates in the wake of a bluff body has shown that the primary vortex shedding can be suppressed for sufficiently long splitter plates. In the present work, we study the problem of a hinged-splitter plate in the wake of a circular cylinder. The splitter plate can rotate about the hinge at the base of the cylinder due to the unsteady fluid forces acting on it, and hence the communication between the two sides of the wake is not totally disrupted as in the rigid splitter plate case. In our study, we investigate this problem in the limit where the stiffness and internal damping associated with the hinge are negligible, and the mass ratio of the splitter plate is small. The experiments show that the splitter plate oscillations increase with Reynolds numbers at low values of Re, and are found to reach a saturation amplitude level at higher Re, Re>4000. This type of saturation amplitude level that appears to continue indefinitely with Re, appears to be related to the fact that there is no structural restoring force, and has been observed previously for transversely oscillating cylinders with no restoring force. In the present case, the saturation tip amplitude level can be up to 0.45D, where D is the cylinder diameter. For this hinged-rigid splitter plate case, it is found that the splitter plate length to cylinder diameter ratio (L/D) is crucial in determining the character and magnitude of the oscillations. For small splitter plate lengths (L/D⩽3.0), the oscillations appear to be nearly periodic with tip amplitudes of about 0.45D nearly independent of L/D. The nondimensional oscillation frequencies (fD/U) on the other hand are found to continuously vary with L/D from fD/U≈0.2 at L/D=1 to fD/U≈0.1 at L/D=3. As the splitter plate length is further increased beyond L/D⩾4.0, the character of the splitter plate oscillations suddenly changes. The oscillations become aperiodic with much smaller amplitudes. In this long splitter plate

  5. High Precision Prediction of Functional Sites in Protein Structures

    PubMed Central

    Buturovic, Ljubomir; Wong, Mike; Tang, Grace W.; Altman, Russ B.; Petkovic, Dragutin

    2014-01-01

    We address the problem of assigning biological function to solved protein structures. Computational tools play a critical role in identifying potential active sites and informing screening decisions for further lab analysis. A critical parameter in the practical application of computational methods is the precision, or positive predictive value. Precision measures the level of confidence the user should have in a particular computed functional assignment. Low precision annotations lead to futile laboratory investigations and waste scarce research resources. In this paper we describe an advanced version of the protein function annotation system FEATURE, which achieved 99% precision and average recall of 95% across 20 representative functional sites. The system uses a Support Vector Machine classifier operating on the microenvironment of physicochemical features around an amino acid. We also compared performance of our method with state-of-the-art sequence-level annotator Pfam in terms of precision, recall and localization. To our knowledge, no other functional site annotator has been rigorously evaluated against these key criteria. The software and predictive models are incorporated into the WebFEATURE service at http://feature.stanford.edu/wf4.0-beta. PMID:24632601

  6. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  7. Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis.

    PubMed

    Lee, M M; Schiefelbein, J

    2001-05-01

    The duplication and divergence of developmental control genes is thought to have driven morphological diversification during the evolution of multicellular organisms. To examine the molecular basis of this process, we analyzed the functional relationship between two paralogous MYB transcription factor genes, WEREWOLF (WER) and GLABROUS1 (GL1), in Arabidopsis. The WER and GL1 genes specify distinct cell types and exhibit non-overlapping expression patterns during Arabidopsis development. Nevertheless, reciprocal complementation experiments with a series of gene fusions showed that WER and GL1 encode functionally equivalent proteins, and their unique roles in plant development are entirely due to differences in their cis-regulatory sequences. Similar experiments with a distantly related MYB gene (MYB2) showed that its product cannot functionally substitute for WER or GL1. Furthermore, an analysis of the WER and GL1 proteins shows that conserved sequences correspond to specific functional domains. These results provide new insights into the evolution of the MYB gene family in Arabidopsis, and, more generally, they demonstrate that novel developmental gene function may arise solely by the modification of cis-regulatory sequences.

  8. DNA and RNA aptamers as modulators of protein function.

    PubMed

    Ulrich, Henning

    2005-03-01

    The SELEX technique (systematic evolution of ligands by exponential enrichment) is a combinatorial library approach in which DNA or RNA molecules are selected by their ability to bind their protein targets with high affinity and specificity. The isolated molecules are referred to as aptamers (from aptus = Latin "to fit"). First, RNA and DNA aptamers were identified that bind to proteins naturally interacting with nucleic acids, or to small organic molecules such as ATP. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands for a wide range of proteins of importance for therapy, and diagnostics. Since these RNA and DNA molecules bind their targets with similar affinities as antibodies, and are able to distinguish between isotypes of an enzyme, aptamers have been also called synthetic antibodies. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as receptors that are only functional in their membrane-bound form, cells, and trypanosomes. RNA aptamers have been expressed in living cells where they inhibit a protein implicated in intracellular signal transduction. The utility of aptamers for in vivo experiments, and diagnostic and therapeutic purposes, is considerably enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Recently, such inhibitors have been evolved for a great variety of targets, including receptors, growth factors, and adhesion molecules implicated in disease. Furthermore, some results were already obtained in animal models and clinical trials. PMID:16787315

  9. The Structure and Function of Non-Collagenous Bone Proteins

    NASA Technical Reports Server (NTRS)

    Hook, Magnus

    1997-01-01

    The long-term goal for this program is to determine the structural and functional relationships of bone proteins and proteins that interact with bone. This information will used to design useful pharmacological compounds that will have a beneficial effect in osteoporotic patients and in the osteoporotic-like effects experienced on long duration space missions. The first phase of this program, funded under a cooperative research agreement with NASA through the Texas Medical Center, aimed to develop powerful recombinant expression systems and purification methods for production of large amounts of target proteins. Proteins expressed in sufficient'amount and purity would be characterized by a variety of structural methods, and made available for crystallization studies. In order to increase the likelihood of crystallization and subsequent high resolution solution of structures, we undertook to develop expression of normal and mutant forms of proteins by bacterial and mammalian cells. In addition to the main goals of this program, we would also be able to provide reagents for other related studies, including development of anti-fibrotic and anti-metastatic therapeutics.

  10. DING proteins: numerous functions, elusive genes, a potential for health.

    PubMed

    Bernier, François

    2013-09-01

    DING proteins, named after their conserved N-terminus, form an overlooked protein family whose members were generally discovered through serendipity. It is characterized by an unusually high sequence conservation, even between distantly related species, and by an outstanding diversity of activities and ligands. They all share a demonstrated capacity to bind phosphate with high affinity or at least a predicted phosphate-binding site. However, DING protein genes are conspicuously absent from databases. The many novel family members identified in recent years have confirmed that DING proteins are ubiquitous not only in animals and plants but probably also in prokaryotes. At the functional level, there is increasing evidence that they participate in many health-related processes such as cancers as well as bacterial (Pseudomonas) and viral (HIV) infections, by mechanisms that are now beginning to be understood. They thus represent potent targets for the development of novel therapeutic approaches, especially against HIV. The few genomic sequences that are now available are starting to give some clues on why DING protein genes and mRNAs are well conserved and difficult to clone. This could open a new era of research, of both fundamental and applied importance. PMID:23743708

  11. Protein side chain conformation predictions with an MMGBSA energy function.

    PubMed

    Gaillard, Thomas; Panel, Nicolas; Simonson, Thomas

    2016-06-01

    The prediction of protein side chain conformations from backbone coordinates is an important task in structural biology, with applications in structure prediction and protein design. It is a difficult problem due to its combinatorial nature. We study the performance of an "MMGBSA" energy function, implemented in our protein design program Proteus, which combines molecular mechanics terms, a Generalized Born and Surface Area (GBSA) solvent model, with approximations that make the model pairwise additive. Proteus is not a competitor to specialized side chain prediction programs due to its cost, but it allows protein design applications, where side chain prediction is an important step and MMGBSA an effective energy model. We predict the side chain conformations for 18 proteins. The side chains are first predicted individually, with the rest of the protein in its crystallographic conformation. Next, all side chains are predicted together. The contributions of individual energy terms are evaluated and various parameterizations are compared. We find that the GB and SA terms, with an appropriate choice of the dielectric constant and surface energy coefficients, are beneficial for single side chain predictions. For the prediction of all side chains, however, errors due to the pairwise additive approximation overcome the improvement brought by these terms. We also show the crucial contribution of side chain minimization to alleviate the rigid rotamer approximation. Even without GB and SA terms, we obtain accuracies comparable to SCWRL4, a specialized side chain prediction program. In particular, we obtain a better RMSD than SCWRL4 for core residues (at a higher cost), despite our simpler rotamer library. Proteins 2016; 84:803-819. © 2016 Wiley Periodicals, Inc.

  12. A minimal sequence code for switching protein structure and function.

    PubMed

    Alexander, Patrick A; He, Yanan; Chen, Yihong; Orban, John; Bryan, Philip N

    2009-12-15

    We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4beta+alpha fold can be transformed into an albumin-binding, 3-alpha fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4beta+alpha to 3-alpha structure can occur via a single amino acid substitution. On one side of the switch point, the 4beta+alpha fold is >90% populated (pH 7.2, 20 degrees C). A single mutation switches the conformation to the 3-alpha fold, which is >90% populated (pH 7.2, 20 degrees C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin. PMID:19923431

  13. Adrenal Mitochondria and Steroidogenesis: From Individual Proteins to Functional Protein Assemblies.

    PubMed

    Midzak, Andrew; Papadopoulos, Vassilios

    2016-01-01

    The adrenal cortex is critical for physiological function as the central site of glucocorticoid and mineralocorticoid synthesis. It possesses a great degree of specialized compartmentalization at multiple hierarchical levels, ranging from the tissue down to the molecular levels. In this paper, we discuss this functionalization, beginning with the tissue zonation of the adrenal cortex and how this impacts steroidogenic output. We then discuss the cellular biology of steroidogenesis, placing special emphasis on the mitochondria. Mitochondria are classically known as the "powerhouses of the cell" for their central role in respiratory adenosine triphosphate synthesis, and attention is given to mitochondrial electron transport, in both the context of mitochondrial respiration and mitochondrial steroid metabolism. Building on work demonstrating functional assembly of large protein complexes in respiration, we further review research demonstrating a role for multimeric protein complexes in mitochondrial cholesterol transport, steroidogenesis, and mitochondria-endoplasmic reticulum contact. We aim to highlight with this review the shift in steroidogenic cell biology from a focus on the actions of individual proteins in isolation to the actions of protein assemblies working together to execute cellular functions. PMID:27524977

  14. Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

    PubMed

    Li, Fei; Liu, Jian; Liu, Nan; Kuhn, Leslie A; Garavito, R Michael; Ferguson-Miller, Shelagh

    2016-05-24

    Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.

  15. Adrenal Mitochondria and Steroidogenesis: From Individual Proteins to Functional Protein Assemblies

    PubMed Central

    Midzak, Andrew; Papadopoulos, Vassilios

    2016-01-01

    The adrenal cortex is critical for physiological function as the central site of glucocorticoid and mineralocorticoid synthesis. It possesses a great degree of specialized compartmentalization at multiple hierarchical levels, ranging from the tissue down to the molecular levels. In this paper, we discuss this functionalization, beginning with the tissue zonation of the adrenal cortex and how this impacts steroidogenic output. We then discuss the cellular biology of steroidogenesis, placing special emphasis on the mitochondria. Mitochondria are classically known as the “powerhouses of the cell” for their central role in respiratory adenosine triphosphate synthesis, and attention is given to mitochondrial electron transport, in both the context of mitochondrial respiration and mitochondrial steroid metabolism. Building on work demonstrating functional assembly of large protein complexes in respiration, we further review research demonstrating a role for multimeric protein complexes in mitochondrial cholesterol transport, steroidogenesis, and mitochondria–endoplasmic reticulum contact. We aim to highlight with this review the shift in steroidogenic cell biology from a focus on the actions of individual proteins in isolation to the actions of protein assemblies working together to execute cellular functions. PMID:27524977

  16. Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling.

    PubMed

    Mchaourab, H S; Oh, K J; Fang, C J; Hubbell, W L

    1997-01-14

    T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

  17. Pulse Dipolar ESR and Protein Superstructures and Function

    NASA Astrophysics Data System (ADS)

    Freed, Jack

    2014-03-01

    Pulse dipolar electron-spin resonance (PDS-ESR) has emerged as a powerful methodology for the study of protein structure and function. This technology, in the form of double quantum coherence (DQC) - ESR and double-electron-electron resonance (DEER) in conjunction with site-directed spin-labeling will be described. It enables the measurement of distances and their distributions in the range of 1-9 nm between pairs of spins labeled at two sites in the protein. Many biological objects can be studied: soluble and membrane proteins, protein complexes, etc. Many sample morphologies are possible: uniform, heterogeneous, etc. thereby permitting a variety of sample types: solutions, liposomes, micelles, bicelles. Concentrations from micromolar to tens of millimolar are amenable, requiring only small amounts of biomolecules. The distances are quite accurate, so a relatively small number of them are sufficient to reveal structures and functional details. Several examples will be shown. The first is defining the protein complexes that mediate bacterial chemotaxis, which is the process whereby cells modulate their flagella-driven motility in response to environmental cues. It relies on a complex sensory apparatus composed of transmembrane receptors, histidine kinases, and coupling proteins. PDS-based models have captured key architectural features of the receptor kinase arrays and the flagellar motor, and their changes in conformation and dynamics that accompany kinase activation and motor switching. Another example will be determining the conformational states and cycling of a membrane transporter, GltPh, which is a homotrimer, in its apo, substrate-bound, and inhibitor-bound, states in membrane vesicles providing insight into its energetics. In a third example the structureless (in solution) proteins alpha-synuclein and tau, which are important in Parkinson's disease and in neurodegeneration will be described and the structures they take on in contact with membranes will be

  18. Polyethyleneimine-modified graphene oxide nanocomposites for effective protein functionalization

    NASA Astrophysics Data System (ADS)

    Weng, Yejing; Jiang, Bo; Yang, Kaiguang; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2015-08-01

    A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions.A facile method to prepare a biocompatible graphene oxide (GO)-based substrate for protein immobilization was developed to overcome the drawbacks of GO, such as the strong electrostatic and hydrophobic interactions which could potentially alter the conformation and biological activity of proteins. The GO was coated with hydrophilic branched polyethyleneimine (BPEI), while Concanavalin A (Con A) as a model lectin protein was employed to fabricate the functionalized composites to evaluate the feasibility of this strategy. The composites exhibit an extremely high binding capacity for glycoproteins (i.e. IgG 538.3 mg g-1), which are superior to other immobilized materials. Moreover, they can work well in 500-fold non-glycoprotein interference and even in complex biological samples. All these data suggest that the GO@BPEI composites will have great potential as scaffolds for proteins fully exerting their biofunctions. Electronic supplementary information (ESI) available: Cell viability assay, enrichment of standard glycoprotein, pretreatment and analysis of real

  19. A novel alignment mechanism employing orthogonal connected multi-layered flexible hinges for both leveling and centering.

    PubMed

    Zhao, Jian; Wang, Hongxi; Gao, Renjing; Hu, Ping; Yang, Yintang

    2012-06-01

    To eliminate the effects of motion coupling for measuring cylindrical work pieces, a novel alignment mechanism integrating functions of both leveling and centering is designed and fabricated by introducing multi-layered orthogonal connected flexible hinges as the key supporting and joining elements. Different from traditional leveling mechanisms with many separate parts fabricated together, all of the flexible hinges were integrated in one three-dimensioned machining part without assembling process, and thus synchronously simplifying the structure and reducing assembly errors. Based on the screw theory, the mathematic model of the proposed alignment mechanism is established for any resolution requirements depending on screw characteristics. A millimeter-sized device is fabricated with the alignment precision of 1.0 μm for centering within the range of ±1 mm and 1 in. for leveling within ±1°. The experiment results are in very close agreement to those obtained by simulation, which validate the feasibility of introducing multi-layered orthogonal flexible hinges in the centering and leveling mechanisms. PMID:22755657

  20. Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.

    PubMed

    Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N; Braus, Gerhard H

    2002-05-14

    The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.

  1. [Peptide synthesis aiming at elucidation and creation of protein functions].

    PubMed

    Futaki, S

    1998-11-01

    The recent development of molecular biology has been elucidating outlines of the cross-talk of biomolecules. The understanding of the function of these biomolecules from the viewpoint of chemistry is now demanded not only for the understanding of biological systems but also for the creation of novel functional molecules. Here two topics are described about peptide synthesis aiming at the elucidation and the creation of protein functions. The first topic is the development of approaches for the synthesis of Tyr (SO3H)-containing peptides. Tyrosine sulfation is one of the most popular protein post-translational modifications. Synthetic peptides are of great help for the elucidation of the biological significance of tyrosine sulfation. We have developed two approaches for the efficient synthesis of tyrosine sulfate [Tyr (SO3H)]-containing peptides. The first approach employs a dimethylformamide-sulfur trioxide (DMF-SO3) complex as a sulfating agent and safety-catch protecting groups for the selective sulfation of tyrosine in the presence of serine. The second approach employs the direct introduction of Tyr(SO3H) into the peptide chain in the form of Fmoc-Tyr(SO3Na) followed by deprotection at 4 degrees C in trifluoroacetic acid. These approaches were successfully applied for the synthesis of cholecystokinin (CCK)-related peptides. The second topic deals with new approaches for the creation of artificial proteins through assembling alpha-helical peptides via selective disulfide or thioether formation. Approaches to assemble individual peptide segments on a peptide template were also developed. Four peptides corresponding to the transmembrane segments of the sodium channel (S4 in repeat I-IV) were assembled on a peptide template to give a protein having ion channel activity with rectification.

  2. Decreased function of survival motor neuron protein impairs endocytic pathways.

    PubMed

    Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S; O'Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C Q; Cook, Steven J; Poulogiannis, George; Atwood, Walter J; Hall, David H; Hart, Anne C

    2016-07-26

    Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death. PMID:27402754

  3. Uncoupling proteins: a complex journey to function discovery.

    PubMed

    Cioffi, Federica; Senese, Rosalba; de Lange, Pieter; Goglia, Fernando; Lanni, Antonia; Lombardi, Assunta

    2009-01-01

    Since their discovery, uncoupling proteins have aroused great interest due to the crucial importance of energy-dissipating system for cellular physiology. The uncoupling effect and the physiological role of UCP1 (the first-described uncoupling protein) are well established. However, the reactions catalyzed by UCP1 homologues (UCPs), and their physiological roles are still under debate, with the literature containing contrasting results. Current hypothesis propose several physiological functions for novel UCPs, such as: (i) attenuation of reactive oxygen species production and protection against oxidative damage, (ii) thermogenic function, although UCPs do not generally seem to affect thermogenesis, UCP3 can be thermogenic under certain conditions, (iii) involvement in fatty acid handling and/or transport, although recent experimental evidence argues against the previously hypothesized role for UCPs in the export of fatty acid anions, (iv) fatty acid hydroperoxide export, although this function, due to the paucity of the experimental evidence, remains hypothetical, (v) Ca(2+) uptake, although results for and against a role in Ca(2+) uptake are still emerging, (vi) a signaling role in pancreatic beta cells, where it attenuates glucose-induced insulin secretion. From the above, it is evident that more research will be needed to establish universally accepted functions for UCPs.

  4. The importance of slow motions for protein functional loops

    NASA Astrophysics Data System (ADS)

    Skliros, Aris; Zimmermann, Michael T.; Chakraborty, Debkanta; Saraswathi, Saras; Katebi, Ataur R.; Leelananda, Sumudu P.; Kloczkowski, Andrzej; Jernigan, Robert L.

    2012-02-01

    Loops in proteins that connect secondary structures such as alpha-helix and beta-sheet, are often on the surface and may play a critical role in some functions of a protein. The mobility of loops is central for the motional freedom and flexibility requirements of active-site loops and may play a critical role for some functions. The structures and behaviors of loops have not been studied much in the context of the whole structure and its overall motions, especially how these might be coupled. Here we investigate loop motions by using coarse-grained structures (Cα atoms only) to solve the motions of the system by applying Lagrange equations with elastic network models to learn about which loops move in an independent fashion and which move in coordination with domain motions, faster and slower, respectively. The normal modes of the system are calculated using eigen-decomposition of the stiffness matrix. The contribution of individual modes and groups of modes is investigated for their effects on all residues in each loop by using Fourier analyses. Our results indicate overall that the motions of functional sets of loops behave in similar ways as the whole structure. But overall only a relatively few loops move in coordination with the dominant slow modes of motion, and these are often closely related to function.

  5. The chaperone like function of the nonhistone protein HMGB1

    SciTech Connect

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-03-08

    -synthetic acetylation for the chaperone function of HMGB1 protein. The presence of an acetyl groups at Lys 2 decreases strongly the stimulating effect of the protein in the stepwise salt dialysis experiment and the same tendency persisted in the dialysis free experiment.

  6. Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension.

    PubMed

    Kumarasamy, Sivarajan; Waghulde, Harshal; Gopalakrishnan, Kathirvel; Mell, Blair; Morgan, Eric; Joe, Bina

    2015-01-01

    Genome-wide association studies (GWAS) have prioritized a transcription factor, nuclear receptor 2 family 2 (NR2F2), as being associated with essential hypertension in humans. Here we provide evidence that validates this association and indicates that Nr2f2 is a genetic determinant of blood pressure (BP). Using the zinc-finger nuclease technology, the generation of a targeted Nr2f2-edited rat model is reported. The resulting gene-edited rats have a 15 bp deletion in exon 2 leading to a five-amino-acid deletion in the hinge region of the mutant Nr2f2 protein. Both systolic and diastolic blood pressures of the Nr2f2(mutant) rats are significantly lower than controls. Because the hinge region of Nr2f2 is required for interaction with Friend of Gata2 (Fog2), protein-protein interaction is examined. Interaction of Nr2f2(mutant) protein with Fog2 is greater than that with the wild-type Nr2f2, indicating that the extent of interaction between these two transcription factors critically influences BP. PMID:25687237

  7. The motions of hinged-barge systems in regular seas

    NASA Astrophysics Data System (ADS)

    Kraemer, David Robert Burke

    Harnessing the oceans' vast, clean, and renewable energy to do useful work is a tempting prospect. For over a century, wave-energy conversion devices have been proposed, but none has emerged as a clearly practical and economical solution. One promising system is the McCabe Wave Pump (MWP), an articulated-barge system consisting of three barges hinged together with a large horizontal plate attached below the central barge. Water pumps are driven by the relative pitching motions of the barges excited by ocean waves. This high-pressure water can be used to produce potable water or electricity. A simulation of the motions of a generic hinged-barge system is developed. The equations of motion are developed so that the nonlinear interactions between the barges are included. The simulation is general so that it can be used to study other hinged-barge systems, such as causeway ferry systems or floating airports. The simulation is used to predict the motions of a scale model that was studied in wave-tank experiments. In the experimental study, it was observed that the plate attached to the central barge acted as a pendulum. It was also observed that the phases of the pitching motions of the barges was such that the motions were enhanced by the pendulum effect at all of the wave periods studied. Hence, the increased angular displacements produced greater relative pitching motions which would lead to higher volume rates of pumped water in the operational system. The numerical simulations are found to predict the pendulum effect. In addition, the theory predicted that the after barge motions were significantly less than those of the forward barge, as was observed in the experimental study. The good agreement between the two data sets gives confidence in the ability of the theory to predict the performance of the MWP prototype. The motions of the MWP prototype in regular ocean waves are predicted by the simulation, and its performance is calculated. By modifying the length of

  8. Protein functionalized micro hydrogel features for cell-surface interaction.

    PubMed

    Bhatnagar, Parijat; Nixon, Alan J; Kim, Il; Kameoka, Jun

    2008-08-01

    Cross-linked hydrogel features have been patterned using subtractive lift-off of polymerized hydrogel film. Projection lithography and oxygen plasma etch was used to pattern parylene C polymer film. Molecular self-assembly of polymerizable monolayer was obtained in solution-phase and acrylamide based hydrogel was polymerized using free-radical polymerization on this substrate. Parylene C film was mechanically lifted-off to remove the blanket hydrogel film and micro hydrogel features (muhf) were obtained attached to the predefined patterns in the range from 1 to 60 mum. The muhf were functionalized with aldehyde functional groups, and proteins were coupled to them using Schiff base chemistry followed by reductive amination. Interaction of mesenchymal stem cells with transforming growth factor-beta 1 (TGF-beta1) functionalized muhf was studied, and TGF-beta1 was found to retain its tumor suppression activity. PMID:18259869

  9. Engineering and Assembly of Protein Modules into Functional Molecular Systems.

    PubMed

    Hirschi, Stephan; Stauffer, Mirko; Harder, Daniel; Müller, Daniel J; Meier, Wolfgang; Fotiadis, Dimitrios

    2016-01-01

    Synthetic biology approaches range from the introduction of unique features into organisms to the assembly of isolated biomacromolecules or synthetic building blocks into artificial biological systems with biomimetic or completely novel functionalities. Simple molecular systems can be based on containers on the nanoscale that are equipped with tailored functional modules for various applications in healthcare, industry or biological and medical research. The concept, or vision, of assembling native or engineered proteins and/or synthetic components as functional modules into molecular systems is discussed. The main focus is laid on the engineering of energizing modules generating chemical energy, transport modules using this energy to translocate molecules between compartments of a molecular system, and catalytic modules (bio-)chemically processing the molecules. Further key aspects of this discourse are possible approaches for the assembly of simple nanofactories and their applications in biotechnology and medical health. PMID:27363367

  10. Protein-protein coupling and its application to functional red cell substitutes.

    PubMed

    Kluger, Ronald; Foot, Jonathan S; Vandersteen, Adelle A

    2010-02-28

    The need for an alternative to red cells for oxygen transport in transfusions has led to the creation of hemoglobin-based oxygen carriers, materials produced by chemical modification or genetic engineering of human or bovine hemoglobin. Modifications of the native proteins are necessitated by the spontaneous dissociation of the functional hemoglobin tetramers (alpha(2)beta(2)) into non-functional alphabeta dimers. Based on clinical observations of hypertension resulting from some of these materials, it was proposed that the stabilized tetramers are sufficiently small to extravasate through blood vessels and scavenge nitric oxide, depleting the endothelium of the signal for smooth muscle relaxation. In order to increase size and minimize extravasation while maintaining structure and function, methods for producing larger entities through protein-protein conjugation were developed. Approaches have included the use of nonspecific reagents that polymerize proteins (e.g., polyglutaraldehyde), conjugation to polyethylene glycol, expression of naturally occurring multimers and the use of selective reagents, which is the focus of this article.

  11. SNAP-25, a Known Presynaptic Protein with Emerging Postsynaptic Functions

    PubMed Central

    Antonucci, Flavia; Corradini, Irene; Fossati, Giuliana; Tomasoni, Romana; Menna, Elisabetta; Matteoli, Michela

    2016-01-01

    A hallmark of synaptic specializations is their dependence on highly organized complexes of proteins that interact with each other. The loss or modification of key synaptic proteins directly affects the properties of such networks, ultimately impacting synaptic function. SNAP-25 is a component of the SNARE complex, which is central to synaptic vesicle exocytosis, and, by directly interacting with different calcium channels subunits, it negatively modulates neuronal voltage-gated calcium channels, thus regulating intracellular calcium dynamics. The SNAP-25 gene has been associated with distinct brain diseases, including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and bipolar disorder, indicating that the protein may act as a shared biological substrate among different “synaptopathies”. The mechanisms by which alterations in SNAP-25 may concur to these psychiatric diseases are still undefined, although alterations in neurotransmitter release have been indicated as potential causative processes. This review summarizes recent work showing that SNAP-25 not only controls exo/endocytic processes at the presynaptic terminal, but also regulates postsynaptic receptor trafficking, spine morphogenesis, and plasticity, thus opening the possibility that SNAP-25 defects may contribute to psychiatric diseases by impacting not only presynaptic but also postsynaptic functions. PMID:27047369

  12. SNAP-25, a Known Presynaptic Protein with Emerging Postsynaptic Functions.

    PubMed

    Antonucci, Flavia; Corradini, Irene; Fossati, Giuliana; Tomasoni, Romana; Menna, Elisabetta; Matteoli, Michela

    2016-01-01

    A hallmark of synaptic specializations is their dependence on highly organized complexes of proteins that interact with each other. The loss or modification of key synaptic proteins directly affects the properties of such networks, ultimately impacting synaptic function. SNAP-25 is a component of the SNARE complex, which is central to synaptic vesicle exocytosis, and, by directly interacting with different calcium channels subunits, it negatively modulates neuronal voltage-gated calcium channels, thus regulating intracellular calcium dynamics. The SNAP-25 gene has been associated with distinct brain diseases, including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and bipolar disorder, indicating that the protein may act as a shared biological substrate among different "synaptopathies". The mechanisms by which alterations in SNAP-25 may concur to these psychiatric diseases are still undefined, although alterations in neurotransmitter release have been indicated as potential causative processes. This review summarizes recent work showing that SNAP-25 not only controls exo/endocytic processes at the presynaptic terminal, but also regulates postsynaptic receptor trafficking, spine morphogenesis, and plasticity, thus opening the possibility that SNAP-25 defects may contribute to psychiatric diseases by impacting not only presynaptic but also postsynaptic functions.

  13. Human PIEZO1 Ion Channel Functions as a Split Protein

    PubMed Central

    Bae, Chilman; Suchyna, Thomas M.; Ziegler, Lynn; Sachs, Frederick; Gottlieb, Philip A.

    2016-01-01

    PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. PMID:26963637

  14. Allosteric function and dysfunction of the prion protein.

    PubMed

    Linden, Rafael; Cordeiro, Yraima; Lima, Luis Mauricio T R

    2012-04-01

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP(C)), its conformational conversion, aggregation and precipitation. We recently proposed that PrP(C) serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP(C), which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP(C) and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP(C), and that allosteric dysfunction of PrP(C) has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP(C), as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.

  15. Growth hormone receptor/binding protein: Physiology and function

    SciTech Connect

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P.

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  16. Structure and functions of plant calcium-dependent protein kinases.

    PubMed

    Klimecka, Maria; Muszyńska, Grazyna

    2007-01-01

    Calcium ions as second messengers play an essential role in many important cellular processes. In plants, transient changes in calcium content in the cytosol (calcium signatures) have been observed during growth, development and under stress conditions. Such diverse functions require many different calcium sensors. One of the largest and most differentiated group of calcium sensors are protein kinases, among them calcium-dependent protein kinases (CDPKs) which were identified only in plants and protists. CDPKs have a regulatory domain which is able to bind calcium ions. For regulation of CDPKs activities not only calcium ions but also specific phospholipids and autophosphorylation are responsible. CDPKs have many different substrates, which reflects the diversity of their functions. Potential protein substrates of CDPK are involved in carbon and nitrogen metabolism, phospholipid synthesis, defense responses, ion and water transport, cytoskeleton organization, transcription and hormone responses. Presently, participation of CDPKs in stress signal transduction pathways (e.g., cold, drought, high salinity, wounding) is intensively studied in many laboratories. An intriguing, but still not fully clarified problem is the cross-talk via CDPKs among different signaling pathways that enables signal integration at different levels and ensure appropriate downstream responses.

  17. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing.

    PubMed

    Mehler, Michaela; Eckert, Carl Elias; Busche, Alena; Kulhei, Jennifer; Michaelis, Jonas; Becker-Baldus, Johanna; Wachtveitl, Josef; Dötsch, Volker; Glaubitz, Clemens

    2015-11-13

    Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.

  18. Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

    PubMed

    Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins. PMID:24136528

  19. MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

    PubMed

    Klett, Javier; Núñez-Salgado, Alfonso; Dos Santos, Helena G; Cortés-Cabrera, Álvaro; Perona, Almudena; Gil-Redondo, Rubén; Abia, David; Gago, Federico; Morreale, Antonio

    2012-09-11

    An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

  20. Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

    PubMed

    Ahir, Bhavesh K; Pratten, Margaret K

    2014-01-01

    Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

  1. The human mannose-binding protein functions as an opsonin

    PubMed Central

    1989-01-01

    The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O- polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms. PMID:2469767

  2. A structurally variable hinged tetrahedron framework from DNA origami.

    PubMed

    Smith, David M; Schüller, Verena; Forthmann, Carsten; Schreiber, Robert; Tinnefeld, Philip; Liedl, Tim

    2011-01-01

    Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffolds as well as a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure's surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT.

  3. A Simplified Lateral Hinge Approach to the Proximal Interphalangeal Joint.

    PubMed

    Bain, Gregory I; McGuire, Duncan T; McGrath, Aleksandra M

    2015-09-01

    Proximal interphalangeal joint replacement is an effective treatment for painful arthritis affecting the joint. However, the complication rate is relatively high, with many of these complications related to soft-tissue imbalance or instability. Volar, dorsal, and lateral approaches have all been described with varying results. We describe a new simplified lateral hinge approach that splits the collateral ligament to provide adequate exposure of the joint. Following insertion of the prosthesis the collateral ligament is simply repaired, side-to-side, which stabilizes the joint. As the central slip, opposite collateral ligament, flexor and extensor tendons have not been violated, early active mobilization without splinting is possible, and the risk of instability, swan-neck, and boutonniere deformity are reduced. The indications, contraindications, surgical technique, and rehabilitation protocol are described. PMID:26230632

  4. Delta3: design and control of a flexure hinge mechanism

    NASA Astrophysics Data System (ADS)

    Bacher, Jean-Philippe; Bottinelli, Stefano; Breguet, Jean-Marc; Clavel, Reymond

    2001-10-01

    In the fields of micro positioning, micromanipulation and micro machining, the required motion precision is continuously increasing. The demand also increases for high dynamic performances (large bandwidth, high closed loop stiffness.). In many cases an inappropriate mechanical structure prevents to achieve these objectives. For example backlash or friction have to be reduced as much as possible. In this paper, we propose backlash-free and friction-free manipulators using flexure hinges and direct drive actuators. A three degrees of freedom (dof) parallel robot (X, Y, Z) that is a transposition in a flexible structure of the Delta robot kinematics is presented. We focus on the design and control of the robot. A simple dynamic model is proposed and compared with measurements. The system is characterized and we propose solutions to improve performances. These solutions are tested on a linear stage.

  5. An alternative RNA polymerase I structure reveals a dimer hinge.

    PubMed

    Kostrewa, Dirk; Kuhn, Claus-D; Engel, Christoph; Cramer, Patrick

    2015-09-01

    RNA polymerase I (Pol I) is the central, 14-subunit enzyme that synthesizes the ribosomal RNA (rRNA) precursor in eukaryotic cells. The recent crystal structure of Pol I at 2.8 Å resolution revealed two novel elements: the `expander' in the active-centre cleft and the `connector' that mediates Pol I dimerization [Engel et al. (2013), Nature (London), 502, 650-655]. Here, a Pol I structure in an alternative crystal form that was solved by molecular replacement using the original atomic Pol I structure is reported. The resulting alternative structure lacks the expander but still shows an expanded active-centre cleft. The neighbouring Pol I monomers form a homodimer with a relative orientation distinct from that observed previously, establishing the connector as a hinge between Pol I monomers.

  6. A Simplified Lateral Hinge Approach to the Proximal Interphalangeal Joint.

    PubMed

    Bain, Gregory I; McGuire, Duncan T; McGrath, Aleksandra M

    2015-09-01

    Proximal interphalangeal joint replacement is an effective treatment for painful arthritis affecting the joint. However, the complication rate is relatively high, with many of these complications related to soft-tissue imbalance or instability. Volar, dorsal, and lateral approaches have all been described with varying results. We describe a new simplified lateral hinge approach that splits the collateral ligament to provide adequate exposure of the joint. Following insertion of the prosthesis the collateral ligament is simply repaired, side-to-side, which stabilizes the joint. As the central slip, opposite collateral ligament, flexor and extensor tendons have not been violated, early active mobilization without splinting is possible, and the risk of instability, swan-neck, and boutonniere deformity are reduced. The indications, contraindications, surgical technique, and rehabilitation protocol are described.

  7. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  8. Emerging and Novel Functions of Complement Protein C1q

    PubMed Central

    Kouser, Lubna; Madhukaran, Shanmuga Priyaa; Shastri, Abhishek; Saraon, Anuvinder; Ferluga, Janez; Al-Mozaini, Maha; Kishore, Uday

    2015-01-01

    Complement protein C1q, the recognition molecule of the classical pathway, performs a diverse range of complement and non-complement functions. It can bind various ligands derived from self, non-self, and altered self and modulate the functions of immune and non-immune cells including dendritic cells and microglia. C1q involvement in the clearance of apoptotic cells and subsequent B cell tolerance is more established now. Recent evidence appears to suggest that C1q plays an important role in pregnancy where its deficiency and dysregulation can have adverse effects, leading to preeclampsia, missed abortion, miscarriage or spontaneous loss, and various infections. C1q is also produced locally in the central nervous system, and has a protective role against pathogens and possible inflammatory functions while interacting with aggregated proteins leading to neurodegenerative diseases. C1q role in synaptic pruning, and thus CNS development, its anti-cancer effects as an immune surveillance molecule, and possibly in aging are currently areas of extensive research. PMID:26175731

  9. Protein meta-functional signatures from combining sequence, structure, evolution, and amino acid property information.

    PubMed

    Wang, Kai; Horst, Jeremy A; Cheng, Gong; Nickle, David C; Samudrala, Ram

    2008-09-26

    Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence-structure-function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

  10. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  11. The Neuroprotective Functions of Transforming Growth Factor Beta Proteins

    PubMed Central

    Dobolyi, Arpád; Vincze, Csilla; Pál, Gabriella; Lovas, Gábor

    2012-01-01

    Transforming growth factor beta (TGF-β) proteins are multifunctional cytokines whose neural functions are increasingly recognized. The machinery of TGF-β signaling, including the serine kinase type transmembrane receptors, is present in the central nervous system. However, the 3 mammalian TGF-β subtypes have distinct distributions in the brain suggesting different neural functions. Evidence of their involvement in the development and plasticity of the nervous system as well as their functions in peripheral organs suggested that they also exhibit neuroprotective functions. Indeed, TGF-β expression is induced following a variety of types of brain tissue injury. The neuroprotective function of TGF-βs is most established following brain ischemia. Damage in experimental animal models of global and focal ischemia was shown to be attenuated by TGF-βs. In addition, support for their neuroprotective actions following trauma, sclerosis multiplex, neurodegenerative diseases, infections, and brain tumors is also accumulating. The review will also describe the potential mechanisms of neuroprotection exerted by TGF-βs including anti-inflammatory, -apoptotic, -excitotoxic actions as well as the promotion of scar formation, angiogenesis, and neuroregeneration. The participation of these mechanisms in the neuroprotective effects of TGF-βs during different brain lesions will also be discussed. PMID:22942700

  12. Design of Hierarchically Cut Hinges for Highly Stretchable and Reconfigurable Metamaterials with Enhanced Strength.

    PubMed

    Tang, Yichao; Lin, Gaojian; Han, Lin; Qiu, Songgang; Yang, Shu; Yin, Jie

    2015-11-25

    Applying hierarchical cuts to thin sheets of elastomer generates super-stretchable and reconfigurable metamaterials, exhibiting highly nonlinear stress-strain behaviors and tunable phononic bandgaps. The cut concept fails on brittle thin sheets due to severe stress concentration in the rotating hinges. By engineering the local hinge shapes and global hierarchical structure, cut-based reconfigurable metamaterials with largely enhanced strength are realized.

  13. Radar Interferometry Detection of Hinge Line Migration on Rutford Ice Stream and Carlson Inlet, Antarctica

    NASA Technical Reports Server (NTRS)

    Rignot, Eric

    1997-01-01

    Satellite synthetic-aperture radar (SAR) Interferometry is employed to map the hinge line, or limit of tidal flexing, of Rutford Ice Stream and Carlson Inlet, Antarctica, and detect its migration between 1992 and 1996. The hinge line is mapped using a model fit from an elastic beam theory.

  14. High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase.

    PubMed

    Kitahara, R; Sareth, S; Yamada, H; Ohmae, E; Gekko, K; Akasaka, K

    2000-10-24

    A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.

  15. Biofield-effect protein-sensor: Plasma functionalization of polyaniline, protein immobilization, and sensing mechanism

    NASA Astrophysics Data System (ADS)

    Cho, Chae-Ryong; Lee, Hyun-Uk; Ahn, Kyun; Jeong, Se-Young; Choi, Jun-Hee; Kim, Jinwoo; Cho, Jiung

    2014-06-01

    We report the fabrication of a biofield-effect protein-sensor (BioFEP) based on atmospheric-pressure plasma (AP) treatment of a conducting polyaniline (PANI) film. Successive H2 and O2 AP (OHAP) treatment generated dominant hydrophilic -OH and O=CO- functional groups on the PANI film surface, which served as strong binding sites to immobilize bovine serum albumin (BSA) protein molecules. The output current changes of the BioFEP as a function of BSA concentration were obtained. The resistance of the OHAP surface could be sensitively increased from 2.5 × 108 Ω to 2.0 × 1012 Ω with increasing BSA concentrations in the range of 0.025-4 μg/ml. The results suggest that the method is a simple and cost-effective tool to determine the concentration of BSA by measuring electrical resistance.

  16. Polyglutamylation: a fine-regulator of protein function? 'Protein Modifications: beyond the usual suspects' review series.

    PubMed

    Janke, Carsten; Rogowski, Krzysztof; van Dijk, Juliette

    2008-07-01

    Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.

  17. What's that gene (or protein)? Online resources for exploring functions of genes, transcripts, and proteins

    PubMed Central

    Hutchins, James R. A.

    2014-01-01

    The genomic era has enabled research projects that use approaches including genome-scale screens, microarray analysis, next-generation sequencing, and mass spectrometry–based proteomics to discover genes and proteins involved in biological processes. Such methods generate data sets of gene, transcript, or protein hits that researchers wish to explore to understand their properties and functions and thus their possible roles in biological systems of interest. Recent years have seen a profusion of Internet-based resources to aid this process. This review takes the viewpoint of the curious biologist wishing to explore the properties of protein-coding genes and their products, identified using genome-based technologies. Ten key questions are asked about each hit, addressing functions, phenotypes, expression, evolutionary conservation, disease association, protein structure, interactors, posttranslational modifications, and inhibitors. Answers are provided by presenting the latest publicly available resources, together with methods for hit-specific and data set–wide information retrieval, suited to any genome-based analytical technique and experimental species. The utility of these resources is demonstrated for 20 factors regulating cell proliferation. Results obtained using some of these are discussed in more depth using the p53 tumor suppressor as an example. This flexible and universally applicable approach for characterizing experimental hits helps researchers to maximize the potential of their projects for biological discovery. PMID:24723265

  18. Use of slurry infiltrated fiber concrete (SIFCON) in hinge regions of earthquake resistant structures

    NASA Astrophysics Data System (ADS)

    Wood, Bryan Thane

    This dissertation reports on an experimental and analytical study of the use of precast slurry infiltrated fiber concrete (SIFCON) flexural hinges to improve the seismic resistance of reinforced concrete moment frames. The main thrust of the research was to investigate how different variables effect the nonlinear, cyclic, flexural behavior of reinforced SIFCON hinges, and to determine how to optimize hinge performance. In addition, a conceptual analysis was performed to evaluate the improvement in seismic resistance from using SIFCON hinges in concrete structures. Seven 10″ wide, 16″ deep, and 26″ long reinforced SIFCON hinges were designed and fabricated, then tested under quasi-static loading. All specimens were fabricated using between 9 and 11%, by volume, Dramix 30/50 fibers, made by the Bekaert Corporation. Grade 60, Grade 75, and ASTM A722 (Dywidag) bars were used, in combination with three different SIFCON compression strengths. Additionally, various end connection details were used in testing three different reinforcing arrangements. It was shown that precast SIFCON hinges can exhibit better performance than reinforced concrete hinges. The maximum curvature ductility achieved was 26.4 over a 4″ inch long interior region of a specimen. The curvature ductility of this hinge specimen, when taken over the full 26 inch hinge length, was 10.5. SIFCON hinges absorb approximately 30% more energy than fiber-reinforced concrete hinges. SIFCON hinge ductility is limited by the ultimate tensile strain of the reinforcing steel. Grade 60 reinforcing resulted in the best hinge behavior seen in testing. Transverse ties may be required to prevent buckling of compression reinforcing. SIFCON flexural stiffness is approximately half that of comparable strength reinforced concrete beams. It was found that SIFCON material behavior is highly variable. Fiber orientation and size effects are the main variables that affect SIFCON behavior. Fabrication technique and skill of

  19. UBXD Proteins: A Family of Proteins with Diverse Functions in Cancer

    PubMed Central

    Rezvani, Khosrow

    2016-01-01

    The UBXD family is a diverse group of UBX (ubiquitin-regulatory X) domain-containing proteins in mammalian cells. Members of this family contain a UBX domain typically located at the carboxyl-terminal of the protein. In contrast to the UBX domain shared by all members of UBXD family, the amino-terminal domains are diverse and appear to carry out different roles in a subcellular localization-dependent manner. UBXD proteins are principally associated with the endoplasmic reticulum (ER), where they positively or negatively regulate the ER-associated degradation machinery (ERAD). The distinct protein interaction networks of UBXD proteins allow them to have specific functions independent of the ERAD pathway in a cell type- and tissue context-dependent manner. Recent reports have illustrated that a number of mammalian members of the UBXD family play critical roles in several proliferation and apoptosis pathways dysregulated in selected types of cancer. This review covers recent advances that elucidate the therapeutic potential of selected members of the UBXD family that can contribute to tumor growth. PMID:27754413

  20. Dynamic circadian protein-protein interaction networks predict temporal organization of cellular functions.

    PubMed

    Wallach, Thomas; Schellenberg, Katja; Maier, Bert; Kalathur, Ravi Kiran Reddy; Porras, Pablo; Wanker, Erich E; Futschik, Matthias E; Kramer, Achim

    2013-03-01

    Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner. PMID:23555304

  1. Origin and evolution of yolk proteins: expansion and functional diversification of large lipid transfer protein superfamily.

    PubMed

    Wu, Long Tao; Hui, Jerome H L; Chu, Ka Hou

    2013-04-01

    Vitellogenin (VTG) and apolipoprotein (APO) play a central role in animal reproduction and lipid circulation, respectively. Although previous studies have examined the structural and functional relationships of these large lipid transfer proteins (LLTPs) from an evolutionary perspective, the mechanism in generating these different families have not been addressed in invertebrates. In this study, the most comprehensive phylogenetic and genomic analysis of the LLTP superfamily genes is carried out. We propose the expansion and diversification of LLTPs in invertebrates are mediated via retrotransposon-mediated duplications, followed by either subfunctionalization or neofunctionalization in different lineages. In agreement with a previous hypothesis, our analysis suggests that all LLTPs originate from a series of duplications of a primitive yolk protein gene similar to VTG. Two early consecutive duplications of the yolk protein genes resulted in the formation of microsomal triglyceride transfer protein (MTP) and the APO gene ancestor. Gains and losses of domains and genes occurred in each of these families in different animal lineages, with MTP becoming truncated. MTP maintained only the components stabilizing the huge lipoprotein particle. Surprisingly, for the first time, two VTG-like protein families were found to independently arise in the lineages of insects. This work consolidates the reconstruction of the evolutionary roadmap of the LLTP superfamily and provides the first mechanistic explanation on the expansion of family members via retrotransposition in invertebrates. PMID:23426435

  2. A First Line of Stress Defense: Small Heat Shock Proteins and their function in protein homeostasis

    PubMed Central

    Haslbeck, Martin; Vierling, Elizabeth

    2015-01-01

    Small heat shock proteins (sHsps) are virtually ubiquitous molecular chaperones that can prevent the irreversible aggregation of denaturing proteins. To maintain protein homeostasis, sHsps complex with a variety of nonnative proteins in an ATP-independent manner and, in the context of the stress response, form a first line of defense against protein aggregation. In vertebrates they act to maintain the clarity of the eye lens, and in humans sHsp mutations are linked to myopathies and neuropathies. Although found in all domains of life, sHsps are quite diverse and have evolved independently in metazoans, plants and fungi. sHsp monomers range in size from approximately 12 to 42 kDa and are defined by a conserved β-sandwich α-crystallin domain, flanked by variable N- and C-terminal sequences. Most sHsps form large oligomeric ensembles with a broad distribution of different, sphere- or barrel like oligomers, with the size and structure of the oligomers dictated by features of the N- and C-termini. The activity of sHsps is regulated by mechanisms that change the equilibrium distribution in tertiary features and/or quaternary structure of the sHsp ensembles. Cooperation and/or coassembly between different sHsps in the same cellular compartment adds an underexplored level of complexity to sHsp structure and function. PMID:25681016

  3. A first line of stress defense: small heat shock proteins and their function in protein homeostasis.

    PubMed

    Haslbeck, Martin; Vierling, Elizabeth

    2015-04-10

    Small heat shock proteins (sHsps) are virtually ubiquitous molecular chaperones that can prevent the irreversible aggregation of denaturing proteins. sHsps complex with a variety of non-native proteins in an ATP-independent manner and, in the context of the stress response, form a first line of defense against protein aggregation in order to maintain protein homeostasis. In vertebrates, they act to maintain the clarity of the eye lens, and in humans, sHsp mutations are linked to myopathies and neuropathies. Although found in all domains of life, sHsps are quite diverse and have evolved independently in metazoans, plants and fungi. sHsp monomers range in size from approximately 12 to 42kDa and are defined by a conserved β-sandwich α-crystallin domain, flanked by variable N- and C-terminal sequences. Most sHsps form large oligomeric ensembles with a broad distribution of different, sphere- or barrel-like oligomers, with the size and structure of the oligomers dictated by features of the N- and C-termini. The activity of sHsps is regulated by mechanisms that change the equilibrium distribution in tertiary features and/or quaternary structure of the sHsp ensembles. Cooperation and/or co-assembly between different sHsps in the same cellular compartment add an underexplored level of complexity to sHsp structure and function.

  4. Expanding the Range of Protein Function at the Far End of the Order-Structure Continuum.

    PubMed

    Burger, Virginia M; Nolasco, Diego O; Stultz, Collin M

    2016-03-25

    The traditional view of the structure-function paradigm is that a protein's function is inextricably linked to a well defined, three-dimensional structure, which is determined by the protein's primary amino acid sequence. However, it is now accepted that a number of proteins do not adopt a unique tertiary structure in solution and that some degree of disorder is required for many proteins to perform their prescribed functions. In this review, we highlight how a number of protein functions are facilitated by intrinsic disorder and introduce a new protein structure taxonomy that is based on quantifiable metrics of a protein's disorder.

  5. Targeting protein function: the expanding toolkit for conditional disruption

    PubMed Central

    Campbell, Amy E.; Bennett, Daimark

    2016-01-01

    A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours. PMID:27574023

  6. Targeting protein function: the expanding toolkit for conditional disruption.

    PubMed

    Campbell, Amy E; Bennett, Daimark

    2016-09-01

    A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.

  7. Associated proteins and renal epithelial Na+ channel function.

    PubMed

    Ismailov, I I; Berdiev, B K; Bradford, A L; Awayda, M S; Fuller, C M; Benos, D J

    1996-01-01

    The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K(amil)i) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K(amil)i following stretch (0.44 +/- 0.12 microM versus 6.9 +/- 1.0 microM). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of alpha bENaC recognized a 70 kDa polypeptide component of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins. PMID:8834119

  8. Intracellular shuttling and mitochondrial function of thioredoxin-interacting protein.

    PubMed

    Saxena, Geetu; Chen, Junqin; Shalev, Anath

    2010-02-01

    The thioredoxin-interacting protein TXNIP is a ubiquitously expressed redox protein that promotes apoptosis. Recently, we found that TXNIP deficiency protects against type 1 and 2 diabetes by inhibiting beta cell apoptosis and maintaining pancreatic beta cell mass, indicating that TXNIP plays a key role in beta cell biology. However, very little is known about the intracellular localization and function of TXNIP, and although TXNIP has been thought to be a cytoplasmic protein, our immunohistochemistry studies in beta cells surprisingly revealed a nuclear TXNIP localization, suggesting that TXNIP may shuttle within the cell. Using immunohistochemistry/confocal imaging and cell fractionation/co-immunoprecipitation, we found that, under physiological conditions, TXNIP is localized primarily in the nucleus of pancreatic beta cells, whereas oxidative stress leads to TXNIP shuttling into the mitochondria. In mitochondria, TXNIP binds to and oxidizes Trx2, thereby reducing Trx2 binding to ASK1 and allowing for ASK1 phosphorylation/activation, resulting in induction of the mitochondrial pathway of apoptosis with cytochrome c release and caspase-3 cleavage. TXNIP overexpression and Trx2 (but not cytosolic Trx1) silencing mimic these effects. Thus, we discovered that TXNIP shuttles between subcellular compartments in response to oxidative stress and identified a novel redox-sensitive mitochondrial TXNIP-Trx2-ASK1 signaling cascade.

  9. Versatile Functions of p53 Protein in Multicellular Organisms

    PubMed Central

    Chumakov, P. M.

    2009-01-01

    The p53 tumor suppressor plays a pivotal role by controlling virtually all processes in the cell. The functions of p53 determine modes of behavior of cells in multicellular organisms and ensure priorities of interests of the organism as a whole above the interests of an individual cell. Multiple signaling pathways of the cell report signals modifying the activities of p53 through numerous connections, ensuring highly selective and gradual regulation of functions that depend on the ongoing events in the cell. The task of p53 is to control the integrity and correctness of all processes in each individual cell and in the organism as a whole. The changes in the activity of p53 depend on the degree of errors or faults, and the effect is directed either toward correction of an imbalance or damage, or, in case of severe damages, leads to the prevention of multiplication of abnormal cells or their death. The strategy of p53 ensures genetic identity of cells and prevents the selection of cells having growth or other advantages. By accomplishing these strategic tasks, p53 may use a wide spectrum of activities. The majority of the activities are due to the ability of p53 to function as a transcription factor, by inducing or repressing different genes. However, p53 can also function as an enzyme, acting as an exonuclease during DNA reparation, or as an adaptor or a regulatory protein, intervening into functions of numerous signaling pathways. It can also act as direct inducer of apoptosis by translocation into mitochondria. Loss of function of the p53 gene occurs in virtually every case of cancer, and deficiency in p53 is an unavoidable prerequisite to the development of malignancies. The functions of p53 play substantial roles in many other pathologies as well as in the aging process. This review is focused on strategies of the p53 gene, demonstrating individual mechanisms underlying its functions. The p53 tumor suppressor plays a pivotal role in multicellular organism by

  10. Impact of C-reactive protein (CRP) on surfactant function

    SciTech Connect

    Li, J.J.; Sanders, R.L.; McAdam, K.P.; Hales, C.A.; Thompson, B.T.; Gelfand, J.A.; Burke, J.F. )

    1989-12-01

    Plasma levels of the acute-phase reactant, C-reactive protein (CRP), increase up to one thousand-fold as a result of trauma or inflammation. CRP binds to phosphorylcholine (PC) in a calcium-ion dependent manner. The structural homology between PC and the major phospholipid component of surfactant, dipalmitoyl phosphatidylcholine (DPPC), led to the present study in which we examined if CRP levels might be increased in patients with adult respiratory distress syndrome (ARDS), and subsequently interfere with surfactant function. Our results showed that CRP levels in the bronchoalveolar fluid (BALF) was increased in patients with ARDS (97.8 +/- 84.2 micrograms/mg total protein vs. 4.04 +/- 2.2 micrograms/mg total protein in normals). Our results show that CRP binds to liposomes containing DPPC and phosphatidylglycerol (PG). As a result of this interaction, CRP inhibits the surface activity of a PG-DPPC mixture when tested with a Wilhelmy surfactometer or with the Enhorning pulsating bubble apparatus. Furthermore, the surface activity of a clinically used surfactant replacement, Surfactant TA (2 mg/ml), was also severely impaired by CRP in a dose-dependent manner (doses used ranging from 24.5 to 1,175 micrograms/ml). In contrast, human serum albumin (HSA) at 500 and 900 micrograms/ml had no inhibitory effect on Surfactant TA surface activity. These results suggest that CRP, although not an initiating insult in ARDS, may contribute to the subsequent abnormalities of surfactant function and thus the pathogenesis of the pulmonary dysfunction seen in ARDS.

  11. Regulation of mitochondrial functions by protein phosphorylation and dephosphorylation.

    PubMed

    Lim, Sangbin; Smith, Kelly R; Lim, Ssang-Taek Steve; Tian, Rong; Lu, Jianrong; Tan, Ming

    2016-01-01

    The mitochondria are d