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Sample records for protein backbone transitions

  1. Ultraviolet Spectroscopy of Protein Backbone Transitions in Aqueous Solution: combined QM and MM Simulations

    PubMed Central

    Jiang, Jun; Abramavicius, Darius; Bulheller, Benjamin M.; Hirst, Jonathan D.; Mukamel, Shaul

    2010-01-01

    A generalized approach combining Quantum Mechanics (QM) and Molecular Mechanics (MM) calculations is developed to simulate the n → π* and π → π* backbone transitions of proteins in aqueous solution. These transitions, which occur in the ultraviolet (UV) at 180–220 nm, provide a sensitive probe for secondary structures. The excitation Hamiltonian is constructed using high level electronic structure calculations of N-methylacetamide (NMA). Its electrostatic fluctuations are modeled using a new algorithm, EHEF, which combines a molecular dynamics (MD) trajectory obtained with a molecular mechanics forcefield, and electronic structures of sampled MD snapshots calculated by QM. The lineshapes and excitation split-tings induced by the electrostatic environment in the experimental UV linear absorption (LA) and circular dichroism (CD) spectra of several proteins in aqueous solution are reproduced by our calculations. The distinct CD features of α-helix and β-sheet protein structures are observed in the simulations and can be assigned to different backbone geometries. The fine structure of the UV spectra is accurately characterized and enables us to identify signatures of secondary structures. PMID:20503991

  2. Vanishing amplitude of backbone dynamics causes a true protein dynamical transition: 2H NMR studies on perdeuterated C-phycocyanin.

    PubMed

    Kämpf, Kerstin; Kremmling, Beke; Vogel, Michael

    2014-03-01

    Using a combination of H2 nuclear magnetic resonance (NMR) methods, we study internal rotational dynamics of the perdeuterated protein C-phycocyanin (CPC) in dry and hydrated states over broad temperature and dynamic ranges with high angular resolution. Separating H2 NMR signals from methyl deuterons, we show that basically all backbone deuterons exhibit highly restricted motion occurring on time scales faster than microseconds. The amplitude of this motion increases when a hydration shell exists, while it decreases upon cooling and vanishes near 175 K. We conclude that the vanishing of the highly restricted motion marks a dynamical transition, which is independent of the time window and of a fundamental importance. This conclusion is supported by results from experimental and computational studies of the proteins myoglobin and elastin. In particular, we argue based on findings in molecular dynamics simulations that the behavior of the highly restricted motion of proteins at the dynamical transition resembles that of a characteristic secondary relaxation of liquids at the glass transition, namely the nearly constant loss. Furthermore, H2 NMR studies on perdeuterated CPC reveal that, in addition to highly restricted motion, small fractions of backbone segments exhibit weakly restricted dynamics when temperature and hydration are sufficiently high. PMID:24730877

  3. Vanishing amplitude of backbone dynamics causes a true protein dynamical transition: H2 NMR studies on perdeuterated C-phycocyanin

    NASA Astrophysics Data System (ADS)

    Kämpf, Kerstin; Kremmling, Beke; Vogel, Michael

    2014-03-01

    Using a combination of H2 nuclear magnetic resonance (NMR) methods, we study internal rotational dynamics of the perdeuterated protein C-phycocyanin (CPC) in dry and hydrated states over broad temperature and dynamic ranges with high angular resolution. Separating H2 NMR signals from methyl deuterons, we show that basically all backbone deuterons exhibit highly restricted motion occurring on time scales faster than microseconds. The amplitude of this motion increases when a hydration shell exists, while it decreases upon cooling and vanishes near 175 K. We conclude that the vanishing of the highly restricted motion marks a dynamical transition, which is independent of the time window and of a fundamental importance. This conclusion is supported by results from experimental and computational studies of the proteins myoglobin and elastin. In particular, we argue based on findings in molecular dynamics simulations that the behavior of the highly restricted motion of proteins at the dynamical transition resembles that of a characteristic secondary relaxation of liquids at the glass transition, namely the nearly constant loss. Furthermore, H2 NMR studies on perdeuterated CPC reveal that, in addition to highly restricted motion, small fractions of backbone segments exhibit weakly restricted dynamics when temperature and hydration are sufficiently high.

  4. Protein-protein docking with backbone flexibility.

    PubMed

    Wang, Chu; Bradley, Philip; Baker, David

    2007-10-19

    Computational protein-protein docking methods currently can create models with atomic accuracy for protein complexes provided that the conformational changes upon association are restricted to the side chains. However, it remains very challenging to account for backbone conformational changes during docking, and most current methods inherently keep monomer backbones rigid for algorithmic simplicity and computational efficiency. Here we present a reformulation of the Rosetta docking method that incorporates explicit backbone flexibility in protein-protein docking. The new method is based on a "fold-tree" representation of the molecular system, which seamlessly integrates internal torsional degrees of freedom and rigid-body degrees of freedom. Problems with internal flexible regions ranging from one or more loops or hinge regions to all of one or both partners can be readily treated using appropriately constructed fold trees. The explicit treatment of backbone flexibility improves both sampling in the vicinity of the native docked conformation and the energetic discrimination between near-native and incorrect models. PMID:17825317

  5. Conformation Dependence of Backbone Geometry in Proteins

    PubMed Central

    Berkholz, Donald S.; Shapovalov, Maxim V.; Dunbrack, Roland L.; Karplus, P. Andrew

    2009-01-01

    Summary Protein structure determination and predictive modeling have long been guided by the paradigm that the peptide backbone has a single, context-independent ideal geometry. Both quantum-mechanics calculations and empirical analyses have shown this is an incorrect simplification in that backbone covalent geometry actually varies systematically as a function of the Φ and Ψ backbone dihedral angles. Here, we use a nonredundant set of ultrahigh-resolution protein structures to define these conformation-dependent variations. The trends have a rational, structural basis that can be explained by avoidance of atomic clashes or optimization of favorable electrostatic interactions. To facilitate adoption of this new paradigm, we have created a conformation-dependent library of covalent bond lengths and bond angles and shown that it has improved accuracy over existing methods without any additional variables to optimize. Protein structures derived both from crystallographic refinement and predictive modeling both stand to benefit from incorporation of the new paradigm. PMID:19836332

  6. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  7. TMAO influence on the backbone of proteins: an oligoglycine model

    PubMed Central

    Hu, Char Y.; Lynch, Gillian C.; Kokubo, Hironori; Pettitt, B. Montgomery

    2009-01-01

    The study of organic osmolytes has been pivotal in demonstrating the role of solvent effects on the protein backbone in the folding process. Whereas a thermodynamic description of the interactions between the protein backbone and osmolyte has been well defined, the structural analysis of the effect of osmolyte on the protein backbone has been incomplete. Therefore, we have carried out simulations of a peptide backbone model, glycine15 in protecting osmolyte TMAO solution in order to determine the effect of the solution structure on the conformation of the peptide backbone. We demonstrate that the models chosen show that the ensemble of backbone structures shifts towards a more collapsed state in TMAO solution as compared to pure water solution. The collapse is consistent with preferential exclusion of the osmolyte caused by unfavorable interactions between osmolyte and peptide backbone. The exclusion is due to strong triplet correlations of osmolyte, water, and peptide backbone. This provides a clear mechanism demonstrating that even a modest concentration of TMAO forces the protein backbone to adopt a more collapsed structure in the absence of sidechain effects. PMID:19790265

  8. A sampling approach for protein backbone fragment conformations.

    PubMed

    Yu, J Y; Zhang, W

    2013-01-01

    In protein structure prediction, backbone fragment bias information can narrow down the conformational space of the whole polypeptide chain significantly. Unlike existing methods that use fragments as building blocks, the paper presents a probabilistic sampling approach for protein backbone torsion angles by modelling angular correlation of (phi, psi) with a directional statistics distribution. Given a protein sequence and secondary structure information, this method samples backbone fragments conformations by using a backtrack sampling algorithm for the hidden Markov model with multiple inputs and a single output. The proposed approach is applied to a fragment library, and some well-known structural motifs are sampled very well on the optimal path. Computational results show that the method can help to obtain native-like backbone fragments conformations. PMID:23777175

  9. Local backbone structure prediction of proteins.

    PubMed

    de Brevern, Alexandre G; Benros, Cristina; Gautier, Romain; Valadié, Héléne; Hazout, Serge; Etchebest, Catherine

    2004-01-01

    A statistical analysis of the PDB structures has led us to define a new set of small 3D structural prototypes called Protein Blocks (PBs). This structural alphabet includes 16 PBs, each one is defined by the (phi, psi) dihedral angles of 5 consecutive residues. The amino acid distributions observed in sequence windows encompassing these PBs are used to predict by a Bayesian approach the local 3D structure of proteins from the sole knowledge of their sequences. LocPred is a software which allows the users to submit a protein sequence and performs a prediction in terms of PBs. The prediction results are given both textually and graphically. PMID:15724288

  10. Increasing protein production by directed vector backbone evolution

    PubMed Central

    2013-01-01

    Recombinant protein production in prokaryotic and eukaryotic organisms was a key enabling technology for the rapid development of industrial and molecular biotechnology. However, despite all progress the improvement of protein production is an ongoing challenge and of high importance for cost-effective enzyme production. With the epMEGAWHOP mutagenesis protocol for vector backbone optimization we report a novel directed evolution based approach to increase protein production levels by randomly introducing mutations in the vector backbone. In the current study we validate the epMEGAWHOP mutagenesis protocol for three different expression systems. The latter demonstrated the general applicability of the epMEGAWHOP method. Cellulase and lipase production was doubled in one round of directed evolution by random mutagenesis of pET28a(+) and pET22b(+) vector backbones. Protease production using the vector pHY300PLK was increased ~4-times with an average of ~1.25 mutations per kb vector backbone. The epMEGAWHOP does not require any rational understanding of the expression machinery and can generally be applied to enzymes, expression vectors and related hosts. epMEGAWHOP is therefore from our point of view a robust, rapid and straight forward alternative for increasing protein production in general and for biotechnological applications. PMID:23890095

  11. Protein-Backbone Thermodynamics across the Membrane Interface.

    PubMed

    Bereau, Tristan; Kremer, Kurt

    2016-07-01

    The thermodynamics of insertion of a protein in a membrane depends on the fine interplay between backbone and side-chain contributions interacting with the lipid environment. Using computer simulations, we probe how different descriptions of the backbone glycyl unit affect the thermodynamics of insertion of individual residues, dipeptides, and entire transmembrane helices. Due to the lack of reference data, we first introduce an efficient methodology to estimate atomistic potential of mean force (PMF) curves from a series of representative and uncorrelated coarse-grained (CG) snapshots. We find strong discrepancies between two CG models, Martini and PLUM, against reference atomistic PMFs and experiments. Atomistic simulations suggest a weak free energy of insertion between water and a POPC membrane for the glycyl unit, in overall agreement with experimental results despite severe assumptions in our calculations. We show that refining the backbone contribution in PLUM significantly improves the PMF of insertion of the WALP16 transmembrane peptide. An improper balance between the glycyl backbone and the attached side chain will lead to energetic artifacts, rationalizing Martini's overstabilization of WALP's adsorbed interfacial state. It illustrates difficulties associated with free-energy-based parametrizations of single-residue models, as the relevant free energy of partitioning used for force-field parametrization does not arise from the entire residue but rather the solvent-accessible chemical groups. PMID:27138459

  12. Determination of backbone nitrogen-nitrogen J correlations in proteins.

    PubMed

    Theis, K; Dingley, A J; Hoffmann, A; Omichinski, J G; Grzesiek, S

    1997-12-01

    Recently, a quantitative J correlation technique has been presented which makes use of homonuclear Hartmann-Hahn cross-polarization (TOCSY) to measure (3)J(C)'(C)' in proteins isotopically enriched with (13)C [Grzesiek, S. and Bax, A. (1997) J. Biomol. NMR, 9, 207-211]. Since homonuclear Hartmann-Hahn is twice as fast as conventional COSY transfer, this method is much less sensitive to transverse relaxation, which is the principal limiting factor in achieving long-range J-coupling correlations in macromolecules. Here we describe a similar experiment which is used to measure(3) J(NN) coupling constants between sequential amide(15) N nuclei in the backbone of ubiquitin. As expected from the low magnetic moment of (15)N, the (3)J(NN) coupling constants are exceedingly small, with values between 0.14 and 0.36 Hz for residues in β-conformations and values below 0.15 Hz for residues in α-conformations. In contrast to what is expected from a Karplus-type dependence on the backbone angle ψ, large differences in the values of(3) J(NN) are observed for a number of residues with very similar backbone ψ angles. A quantitative description of statistical and systematic errors, in particular of relaxation effects during the TOCSY transfer, shows that these differences are highly significant. PMID:20859784

  13. Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

    SciTech Connect

    Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.; Machius, Mischa; Szyperski, Thomas; Kuhlman, Brian

    2015-10-15

    Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helix bundle protein. Only small perturbations to the backbone, 12 {angstrom}, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 {angstrom}).

  14. Statistical mechanics of protein allostery: Roles of backbone and side-chain structural fluctuations

    NASA Astrophysics Data System (ADS)

    Itoh, Kazuhito; Sasai, Masaki

    2011-03-01

    A statistical mechanical model of allosteric transition of proteins is developed by extending the structure-based model of protein folding to cases that a protein has two different native conformations. Partition function is calculated exactly within the model and free-energy surfaces associated with allostery are derived. In this paper, the model of allosteric transition proposed in a previous paper [Proc. Natl. Acad. Sci. U.S.A 134, 7775 (2010)] is reformulated to describe both fluctuation in side-chain configurations and that in backbone structures in a balanced way. The model is applied to example proteins, Ras, calmodulin, and CheY: Ras undergoes the allosteric transition between guanosine diphosphate (GDP)-bound and guanosine triphosphate (GTP)-bound forms, and the model results show that the GDP-bound form is stabilized enough to prevent unnecessary signal transmission, but the conformation in the GTP-bound state bears large fluctuation in side-chain configurations, which may help to bind multiple target proteins for multiple pathways of signaling. The calculated results of calmodulin show the scenario of sequential ordering in Ca2 + binding and the associated allosteric conformational change, which are realized though the sequential appearing of pre-existing structural fluctuations, i.e., fluctuations to show structures suitable to bind Ca2 + before its binding. Here, the pre-existing fluctuations to accept the second and third Ca2 + ions are dominated by the side-chain fluctuation. In CheY, the calculated side-chain fluctuation of Tyr106 is coordinated with the backbone structural change in the β4-α4 loop, which explains the pre-existing Y-T coupling process in this protein. Ability of the model to explain allosteric transitions of example proteins supports the view that the large entropic effects lower the free-energy barrier of allosteric transition.

  15. A Native to Amyloidogenic Transition Regulated by a Backbone Trigger

    SciTech Connect

    Eakin,C.; Berman, A.; Miranker, A.

    2006-01-01

    Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by beta-2-microglobulin (beta2m) in dialysis-related amyloidosis. Access of beta2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a beta2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamic and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type beta2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.

  16. Monitoring Backbone Hydrogen-Bond Formation in β-Barrel Membrane Protein Folding.

    PubMed

    Raschle, Thomas; Rios Flores, Perla; Opitz, Christian; Müller, Daniel J; Hiller, Sebastian

    2016-05-10

    β-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire β-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale. PMID:27062600

  17. Trimethylamine Ν-oxide Influence on the Backbone of Proteins: An Oligoglycine Model

    SciTech Connect

    Hu, Char Y.; Lynch, Gillian C.; Kokubo, Hironori; Pettitt, Bernard M.

    2010-02-15

    The study of organic osmolytes has been pivotal in demonstrating the role of solvent effects on the protein backbone in the folding process. Although a thermodynamic description of the interactions between the protein backbone and osmolyte has been well defined, the structural analysis of the effect of osmolyte on the protein backbone has been incomplete. Therefore, we have performed simulations of a peptide backbone model, glycine₁₅, in protecting osmolyte trimethylamine Ν-oxide (TMAO) solution, in order to determine the effect of the solution structure on the conformation of the peptide backbone. We show that the models chosen show that the ensemble of backbone structures shifts toward a more collapsed state in TMAO solution as compared with pure water solution. The collapse is consistent with preferential exclusion of the osmolyte caused by unfavorable interactions between osmolyte and peptide backbone. The exclusion is caused by strong triplet correlations of osmolyte, water, and peptide backbone. This provides a clear mechanism showing that even a modest concentration of TMAO forces the protein backbone to adopt a more collapsed structure in the absence of side chain effects.

  18. Computation-Guided Backbone Grafting of a Discontinuous Motif onto a Protein Scaffold

    SciTech Connect

    Azoitei, Mihai L.; Correia, Bruno E.; Ban, Yih-En Andrew; Carrico, Chris; Kalyuzhniy, Oleksandr; Chen, Lei; Schroeter, Alexandria; Huang, Po-Ssu; McLellan, Jason S.; Kwong, Peter D.; Baker, David; Strong, Roland K.; Schief, William R.

    2012-02-07

    The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.

  19. Backbone additivity in the transfer model of protein solvation

    PubMed Central

    Hu, Char Y; Kokubo, Hironori; Lynch, Gillian C; Bolen, D Wayne; Pettitt, B Montgomery

    2010-01-01

    The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (ΔGtr) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N-oxide (TMAO), were calculated from simulations of all atom models, and ΔGtr values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of −54 cal/mol/M compares quite favorably with −43 cal/mol/M determined experimentally. PMID:20306490

  20. Backbone Additivity in the Transfer Model of Protein Solvation

    SciTech Connect

    Hu, Char Y.; Kokubo, Hironori; Lynch, Gillian C.; Bolen, D Wayne; Pettitt, Bernard M.

    2010-05-01

    The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (ΔGtr) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N-oxide (TMAO), were calculated from simulations of all atom models, and ΔGtr values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of –54 cal/mol/Mcompares quite favorably with –43 cal/mol/M determined experimentally.

  1. A new default restraint library for the protein backbone in Phenix: a conformation-dependent geometry goes mainstream

    PubMed Central

    Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.; Karplus, P. Andrew

    2016-01-01

    Chemical restraints are a fundamental part of crystallographic protein structure refinement. In response to mounting evidence that conventional restraints have shortcomings, it has previously been documented that using backbone restraints that depend on the protein backbone conformation helps to address these shortcomings and improves the performance of refinements [Moriarty et al. (2014 ▸), FEBS J. 281, 4061–4071]. It is important that these improvements be made available to all in the protein crystallography community. Toward this end, a change in the default geometry library used by Phenix is described here. Tests are presented showing that this change will not generate increased numbers of outliers during validation, or deposition in the Protein Data Bank, during the transition period in which some validation tools still use the conventional restraint libraries. PMID:26894545

  2. Modeling (15)N NMR chemical shift changes in protein backbone with pressure.

    PubMed

    La Penna, Giovanni; Mori, Yoshiharu; Kitahara, Ryo; Akasaka, Kazuyuki; Okamoto, Yuko

    2016-08-28

    Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence. PMID:27586953

  3. Reconstruction of protein backbones from the BriX collection of canonical protein fragments.

    PubMed

    Baeten, Lies; Reumers, Joke; Tur, Vicente; Stricher, François; Lenaerts, Tom; Serrano, Luis; Rousseau, Frederic; Schymkowitz, Joost

    2008-05-01

    As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures. PMID:18483555

  4. Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain

    PubMed Central

    Schumann, Frank H.; Varadan, Ranjani; Tayakuniyil, Praveen P.; Grossman, Jennifer H.; Camarero, Julio A.; Fushman, David

    2015-01-01

    Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding. PMID:25905098

  5. Folding a protein by discretizing its backbone torsional dynamics

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    1999-05-01

    The aim of this work is to provide a coarse codification of local conformational constraints associated with each folding motif of a peptide chain in order to obtain a rough solution to the protein folding problem. This is accomplished by implementing a discretized version of the soft-mode dynamics on a personal computer (PC). Our algorithm mimics a parallel process as it evaluates concurrent folding possibilities by pattern recognition. It may be implemented in a PC as a sequence of perturbation-translation-renormalization (p-t-r) cycles performed on a matrix of local topological constraints (LTM). This requires suitable representational tools and a periodic quenching of the dynamics required for renormalization. We introduce a description of the peptide chain based on a local discrete variable the values of which label the basins of attraction of the Ramachandran map for each residue. Thus, the local variable indicates the basin in which the torsional coordinates of each residue lie at a given time. In addition, a coding of local topological constraints associated with each secondary and tertiary structural motif is introduced. Our treatment enables us to adopt a computation time step of 81 ps, a value far larger than hydrodynamic drag time scales. Folding pathways are resolved as transitions between patterns of locally encoded structural signals that change within the 10 μs-100 ms time scale range. These coarse folding pathways are generated by the periodic search for structural patterns in the time-evolving LTM. Each pattern is recorded as a contact matrix, an operation subject to a renormalization feedback loop. The validity of our approach is tested vis-a-vis experimentally-probed folding pathways eventually generating tertiary interactions in proteins which recover their active structure under in vitro renaturation conditions. As an illustration, we focus on determining significant folding intermediates and late kinetic bottlenecks that occur within the

  6. Using Excel To Study The Relation Between Protein Dihedral Angle Omega And Backbone Length

    NASA Astrophysics Data System (ADS)

    Shew, Christopher; Evans, Samari; Tao, Xiuping

    How to involve the uninitiated undergraduate students in computational biophysics research? We made use of Microsoft Excel to carry out calculations of bond lengths, bond angles and dihedral angles of proteins. Specifically, we studied protein backbone dihedral angle omega by examining how its distribution varies with the length of the backbone length. It turns out Excel is a respectable tool for this task. An ordinary current-day desktop or laptop can handle the calculations for midsized proteins in just seconds. Care has to be taken to enter the formulas for the spreadsheet column after column to minimize the computing load. Supported in part by NSF Grant #1238795.

  7. Nano-Scale Alignment of Proteins on a Flexible DNA Backbone

    PubMed Central

    Nojima, Tatsuya; Konno, Hiroki; Kodera, Noriyuki; Seio, Kohji; Taguchi, Hideki; Yoshida, Masasuke

    2012-01-01

    Nano-scale alignment of several proteins with freedom of motion is equivalent to an enormous increase in effective local concentration of proteins and will enable otherwise impossible weak and/or cooperative associations between them or with their ligands. For this purpose, a DNA backbone made of six oligodeoxynucleotide (ODN) chains is designed in which five double-stranded segments are connected by four single-stranded flexible linkers. A desired protein with an introduced cysteine is connected covalently to the 5′-end of azido-ODN by catalyst-free click chemistry. Then, six protein-ODN conjugates are assembled with their complementary nucleotide sequences into a single multi-protein-DNA complex, and six proteins are aligned along the DNA backbone. Flexible alignment of proteins is directly observed by high-speed AFM imaging, and association of proteins with weak interaction is demonstrated by fluorescence resonance energy transfer between aligned proteins. PMID:23300700

  8. A Novel Method for Sampling Alpha-Helical Protein Backbones

    DOE R&D Accomplishments Database

    Fain, Boris; Levitt, Michael

    2001-01-01

    We present a novel technique of sampling the configurations of helical proteins. Assuming knowledge of native secondary structure, we employ assembly rules gathered from a database of existing structures to enumerate the geometrically possible 3-D arrangements of the constituent helices. We produce a library of possible folds for 25 helical protein cores. In each case the method finds significant numbers of conformations close to the native structure. In addition we assign coordinates to all atoms for 4 of the 25 proteins. In the context of database driven exhaustive enumeration our method performs extremely well, yielding significant percentages of structures (0.02%--82%) within 6A of the native structure. The method's speed and efficiency make it a valuable contribution towards the goal of predicting protein structure.

  9. Automated Real-Space Refinement of Protein Structures Using a Realistic Backbone Move Set

    PubMed Central

    Haddadian, Esmael J.; Gong, Haipeng; Jha, Abhishek K.; Yang, Xiaojing; DeBartolo, Joe; Hinshaw, James R.; Rice, Phoebe A.; Sosnick, Tobin R.; Freed, Karl F.

    2011-01-01

    Crystals of many important biological macromolecules diffract to limited resolution, rendering accurate model building and refinement difficult and time-consuming. We present a torsional optimization protocol that is applicable to many such situations and combines Protein Data Bank-based torsional optimization with real-space refinement against the electron density derived from crystallography or cryo-electron microscopy. Our method converts moderate- to low-resolution structures at initial (e.g., backbone trace only) or late stages of refinement to structures with increased numbers of hydrogen bonds, improved crystallographic R-factors, and superior backbone geometry. This automated method is applicable to DNA-binding and membrane proteins of any size and will aid studies of structural biology by improving model quality and saving considerable effort. The method can be extended to improve NMR and other structures. Our backbone score and its sequence profile provide an additional standard tool for evaluating structural quality. PMID:21843481

  10. Backbone Solution Structures of Proteins Using Residual Dipolar Couplings: Application to a Novel Structural Genomics Target

    PubMed Central

    Valafar, H.; Mayer, K. L.; Bougault, C. M.; LeBlond, P. D.; Jenney, F. E.; Brereton, P. S.; Adams, M.W.W.; Prestegard, J.H.

    2006-01-01

    Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all side chains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the side chains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins that suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods. PMID:15704012

  11. Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

    PubMed

    Lo, Ryan H; Kroncke, Brett M; Solomon, Tsega L; Columbus, Linda

    2014-10-01

    The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins. PMID:25296323

  12. On the relationship between NMR-derived amide order parameters and protein backbone entropy changes

    PubMed Central

    Sharp, Kim A.; O’Brien, Evan; Kasinath, Vignesh; Wand, A. Joshua

    2015-01-01

    Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O2NH) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O2NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O2axis. A calibration curve for backbone entropy vs. O2NH is developed which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O2NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, e.g. upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O2axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. PMID:25739366

  13. A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens.

    PubMed

    Borders, C L; Broadwater, J A; Bekeny, P A; Salmon, J E; Lee, A S; Eldridge, A M; Pett, V B

    1994-04-01

    We propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a "structural" arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase II, Arg 31 in Streptomyces rubiginosus xylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the "mantle" (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV-1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8003972

  14. On the satisfaction of backbone-carbonyl lone pairs of electrons in protein structures.

    PubMed

    Bartlett, Gail J; Woolfson, Derek N

    2016-04-01

    Protein structures are stabilized by a variety of noncovalent interactions (NCIs), including the hydrophobic effect, hydrogen bonds, electrostatic forces and van der Waals' interactions. Our knowledge of the contributions of NCIs, and the interplay between them remains incomplete. This has implications for computational modeling of NCIs, and our ability to understand and predict protein structure, stability, and function. One consideration is the satisfaction of the full potential for NCIs made by backbone atoms. Most commonly, backbone-carbonyl oxygen atoms located within α-helices and β-sheets are depicted as making a single hydrogen bond. However, there are two lone pairs of electrons to be satisfied for each of these atoms. To explore this, we used operational geometric definitions to generate an inventory of NCIs for backbone-carbonyl oxygen atoms from a set of high-resolution protein structures and associated molecular-dynamics simulations in water. We included more-recently appreciated, but weaker NCIs in our analysis, such as n→π* interactions, Cα-H bonds and methyl-H bonds. The data demonstrate balanced, dynamic systems for all proteins, with most backbone-carbonyl oxygen atoms being satisfied by two NCIs most of the time. Combinations of NCIs made may correlate with secondary structure type, though in subtly different ways from traditional models of α- and β-structure. In addition, we find examples of under- and over-satisfied carbonyl-oxygen atoms, and we identify both sequence-dependent and sequence-independent secondary-structural motifs in which these reside. Our analysis provides a more-detailed understanding of these contributors to protein structure and stability, which will be of use in protein modeling, engineering and design. PMID:26833776

  15. Backrub-like backbone simulation recapitulates natural protein conformational variability and improves mutant side-chain prediction

    PubMed Central

    Smith, Colin A.; Kortemme, Tanja

    2008-01-01

    Summary Incorporation of effective backbone sampling into protein simulation and design is an important step in increasing the accuracy of computational protein modeling. Recent analysis of high-resolution crystal structures has suggested a new model, termed backrub, to describe localized, hinge-like alternative backbone and side chain conformations observed in the crystal lattice. The model involves internal backbone rotations about axes between Cα atoms. Based on this observation, we have implemented a backrub-inspired sampling method in the Rosetta structure prediction and design program. We evaluate this model of backbone flexibility using three different tests. First, we show that Rosetta backrub simulations recapitulate the correlation between backbone and side-chain conformations in the high-resolution crystal structures upon which the model was based. As a second test of backrub sampling, we show that backbone flexibility improves the accuracy of predicting point-mutant side chain conformations over fixed backbone rotameric sampling alone. Finally, we show that backrub sampling of triosephosphate isomerase loop 6 can capture the ms/µs oscillation between the open and closed states observed in solution. Our results suggest that backrub sampling captures a sizable fraction of localized conformational changes that occur in natural proteins. Application of this simple model of backbone motions may significantly improve both protein design and atomistic simulations of localized protein flexibility. PMID:18547585

  16. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB.

    PubMed

    Maier, James A; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E; Simmerling, Carlos

    2015-08-11

    Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements. PMID:26574453

  17. Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone

    PubMed Central

    Doucet, Christine M.; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

    2015-01-01

    Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests

  18. The magnitude of the backbone conformational entropy change in protein folding.

    PubMed

    D'Aquino, J A; Gómez, J; Hilser, V J; Lee, K H; Amzel, L M; Freire, E

    1996-06-01

    The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high-sensitivity microcalorimetric analysis. Each pair of mutants differed only by having alanine or glycine at a specific solvent-exposed position under conditions in which the differences in stability could be attributed to differences in the conformational entropy of the unfolded state. The mutants studied were characterized by different stabilities but had identical heat capacity changes of unfolding (delta Cp), identical solvent-related entropies of unfolding (delta Ssolv), and identical enthalpies of unfolding (delta H) at equivalent temperatures. Accordingly, the differences in stability between the different mutants could be attributed to differences in conformational entropy. The computational studies were aimed at generating the energy profile of backbone conformations as a function of the main chain dihedral angles phi and phi. The energy profiles permit a direct calculation of the probability distribution of different conformers and therefore of the conformational entropy of the backbone. The experimental results presented in this paper indicate that the presence of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 +/- 0.2 cal/K. mol with respect to that of glycine, consistent with a 3.4-fold reduction in the number of allowed conformations in the alanine-containing peptides. Similar results were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side chain had only a small effect as long as the side chains were unbranched at position beta. A further reduction with respect to Ala of only 0.61 and 0.81 cal/K. mol in the backbone

  19. Predicting Disease-Related Proteins Based on Clique Backbone in Protein-Protein Interaction Network

    PubMed Central

    Yang, Lei; Zhao, Xudong; Tang, Xianglong

    2014-01-01

    Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases. PMID:25013377

  20. NMR Backbone Assignment of Large Proteins by Using (13) Cα -Only Triple-Resonance Experiments.

    PubMed

    Wei, Qingtao; Chen, Jiajing; Mi, Juan; Zhang, Jiahai; Ruan, Ke; Wu, Jihui

    2016-07-01

    Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical-shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the (13) Cα nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical-shift displacements, we correctly identify the fidelity of approximately 92 % cross-peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross-peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue-by-residue study of the backbone structure and dynamics of large proteins. PMID:27276173

  1. NMR Structure Determination for Larger Proteins Using Backbone-Only Data

    PubMed Central

    Raman, Srivatsan; Lange, Oliver F.; Rossi, Paolo; Tyka, Michael; Wang, Xu; Aramini, James; Liu, Gaohua; Ramelot, Theresa; Eletsky, Alexander; Szyperski, Thomas; Kennedy, Michael; Prestegard, James; Montelione, Gaetano T.; Baker, David

    2010-01-01

    Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without NMR information on the sidechains for proteins up to 25 kDa by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modelling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search towards the lowest energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kDa, and should enable routine NMR structure determination for larger proteins. PMID:20133520

  2. Robust Chemical Synthesis of Membrane Proteins through a General Method of Removable Backbone Modification.

    PubMed

    Zheng, Ji-Shen; He, Yao; Zuo, Chao; Cai, Xiao-Ying; Tang, Shan; Wang, Zhipeng A; Zhang, Long-Hua; Tian, Chang-Lin; Liu, Lei

    2016-03-16

    Chemical protein synthesis can provide access to proteins with post-translational modifications or site-specific labelings. Although this technology is finding increasing applications in the studies of water-soluble globular proteins, chemical synthesis of membrane proteins remains elusive. In this report, a general and robust removable backbone modification (RBM) method is developed for the chemical synthesis of membrane proteins. This method uses an activated O-to-N acyl transfer auxiliary to install in the Fmoc solid-phase peptide synthesis process a RBM group with switchable reactivity toward trifluoroacetic acid. The method can be applied to versatile membrane proteins because the RBM group can be placed at any primary amino acid. With RBM, the membrane proteins and their segments behave almost as if they were water-soluble peptides and can be easily handled in the process of ligation, purification, and mass characterizations. After the full-length protein is assembled, the RBM group can be readily removed by trifluoroacetic acid. The efficiency and usefulness of the new method has been demonstrated by the successful synthesis of a two-transmembrane-domain protein (HCV p7 ion channel) with site-specific isotopic labeling and a four-transmembrane-domain protein (multidrug resistance transporter EmrE). This method enables practical synthesis of small- to medium-sized membrane proteins or membrane protein domains for biochemical and biophysical studies. PMID:26943264

  3. Direct Observation of the Intrinsic Backbone Torsional Mobility of Disordered Proteins.

    PubMed

    Jain, Neha; Narang, Dominic; Bhasne, Karishma; Dalal, Vijit; Arya, Shruti; Bhattacharya, Mily; Mukhopadhyay, Samrat

    2016-08-23

    The fundamental backbone dynamics of unfolded proteins arising due to intrinsic ϕ-ψ dihedral angle fluctuations dictate the course of protein folding, binding, assembly, and function. These internal fluctuations are also critical for protein misfolding associated with a range of human diseases. However, direct observation and unambiguous assignment of this inherent dynamics in chemically denatured proteins is extremely challenging due to various experimental limitations. To directly map the backbone torsional mobility in the ϕ-ψ dihedral angle space, we used a model intrinsically disordered protein, namely, α-synuclein, that adopts an expanded state under native conditions. We took advantage of nonoccurrence of tryptophan in α-synuclein and created a number of single-tryptophan variants encompassing the entire polypeptide chain. We then utilized highly sensitive picosecond time-resolved fluorescence depolarization measurements that allowed us to discern the site-specific torsional relaxation at a low protein concentration under physiological conditions. For all the locations, the depolarization kinetics exhibited two well-separated rotational-correlation-time components. The shorter, subnanosecond component arises due to the local mobility of the indole side chain, whereas the longer rotational-correlation-time component (1.37 ± 0.15 ns), independent of global tumbling, represents a characteristic timescale for short-range conformational exchange in the ϕ-ψ dihedral space. This correlation time represents an intrinsic timescale for torsional relaxation and is independent of position, which is expected for an extended polypeptide chain having little or no propensity to form persistent structures. We were also able to capture this intrinsic timescale at the N-terminal unstructured domain of the prion protein. Our estimated timescale of the segmental mobility is similar to that of unfolded proteins studied by nuclear magnetic resonance in conjunction with

  4. Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

    PubMed Central

    Cozzi, Roberta; Malito, Enrico; Lazzarin, Maddalena; Nuccitelli, Annalisa; Castagnetti, Andrea; Bottomley, Matthew J.; Margarit, Immaculada; Maione, Domenico; Rinaudo, C. Daniela

    2015-01-01

    Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization. PMID:25942637

  5. Conformation-dependent backbone geometry restraints set a new standard for protein crystallographic refinement

    SciTech Connect

    Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.; Karplus, P. Andrew

    2014-06-17

    Ideal values of bond angles and lengths used as external restraints are crucial for the successful refinement of protein crystal structures at all but the highest of resolutions. The restraints in common usage today have been designed based on the assumption that each type of bond or angle has a single ideal value independent of context. However, recent work has shown that the ideal values are, in fact, sensitive to local conformation, and as a first step toward using such information to build more accurate models, ultra-high resolution protein crystal structures have been used to derive a conformation-dependent library (CDL) of restraints for the protein backbone (Berkholz et al. 2009. Structure. 17, 1316). Here, we report the introduction of this CDL into the Phenix package and the results of test refinements of thousands of structures across a wide range of resolutions. These tests show that use of the conformation dependent library yields models that have substantially better agreement with ideal main-chain bond angles and lengths and, on average, a slightly enhanced fit to the X-ray data. No disadvantages of using the backbone CDL are apparent. In Phenix usage of the CDL can be selected by simply specifying the cdl=True option. This successful implementation paves the way for further aspects of the context-dependence of ideal geometry to be characterized and applied to improve experimental and predictive modelling accuracy.

  6. Conformation-dependent backbone geometry restraints set a new standard for protein crystallographic refinement

    DOE PAGESBeta

    Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.; Karplus, P. Andrew

    2014-06-17

    Ideal values of bond angles and lengths used as external restraints are crucial for the successful refinement of protein crystal structures at all but the highest of resolutions. The restraints in common usage today have been designed based on the assumption that each type of bond or angle has a single ideal value independent of context. However, recent work has shown that the ideal values are, in fact, sensitive to local conformation, and as a first step toward using such information to build more accurate models, ultra-high resolution protein crystal structures have been used to derive a conformation-dependent library (CDL)more » of restraints for the protein backbone (Berkholz et al. 2009. Structure. 17, 1316). Here, we report the introduction of this CDL into the Phenix package and the results of test refinements of thousands of structures across a wide range of resolutions. These tests show that use of the conformation dependent library yields models that have substantially better agreement with ideal main-chain bond angles and lengths and, on average, a slightly enhanced fit to the X-ray data. No disadvantages of using the backbone CDL are apparent. In Phenix usage of the CDL can be selected by simply specifying the cdl=True option. This successful implementation paves the way for further aspects of the context-dependence of ideal geometry to be characterized and applied to improve experimental and predictive modelling accuracy.« less

  7. The effect of chirality and steric hindrance on intrinsic backbone conformational propensities: tools for protein design.

    PubMed

    Childers, Matthew Carter; Towse, Clare-Louise; Daggett, Valerie

    2016-07-01

    The conformational propensities of amino acids are an amalgamation of sequence effects, environmental effects and underlying intrinsic behavior. Many have attempted to investigate neighboring residue effects to aid in our understanding of protein folding and improve structure prediction efforts, especially with respect to difficult to characterize states, such as disordered or unfolded states. Host-guest peptide series are a useful tool in examining the propensities of the amino acids free from the surrounding protein structure. Here, we compare the distributions of the backbone dihedral angles (φ/ψ) of the 20 proteogenic amino acids in two different sequence contexts using the AAXAA and GGXGG host-guest pentapeptide series. We further examine their intrinsic behaviors across three environmental contexts: water at 298 K, water at 498 K, and 8 M urea at 298 K. The GGXGG systems provide the intrinsic amino acid propensities devoid of any conformational context. The alanine residues in the AAXAA series enforce backbone chirality, thereby providing a model of the intrinsic behavior of amino acids in a protein chain. Our results show modest differences in φ/ψ distributions due to the steric constraints of the Ala side chains, the magnitudes of which are dependent on the denaturing conditions. One of the strongest factors modulating φ/ψ distributions was the protonation of titratable side chains, and the largest differences observed were in the amino acid propensities for the rarely sampled αL region. PMID:27284086

  8. Conformation-dependent backbone geometry restraints set a new standard for protein crystallographic refinement

    PubMed Central

    Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.; Karplus, P. Andrew

    2014-01-01

    Ideal values of bond angles and lengths used as external restraints are crucial for the successful refinement of protein crystal structures at all but the highest of resolutions. The restraints in common usage today have been designed based on the assumption that each type of bond or angle has a single ideal value independent of context. However, recent work has shown that the ideal values are, in fact, sensitive to local conformation, and as a first step toward using such information to build more accurate models, ultra-high resolution protein crystal structures have been used to derive a conformation-dependent library (CDL) of restraints for the protein backbone (Berkholz et al. 2009. Structure. 17, 1316). Here, we report the introduction of this CDL into the Phenix package and the results of test refinements of thousands of structures across a wide range of resolutions. These tests show that use of the conformation dependent library yields models that have substantially better agreement with ideal main-chain bond angles and lengths and, on average, a slightly enhanced fit to the X-ray data. No disadvantages of using the backbone CDL are apparent. In Phenix usage of the CDL can be selected by simply specifying the cdl=True option. This successful implementation paves the way for further aspects of the context-dependence of ideal geometry to be characterized and applied to improve experimental and predictive modelling accuracy. PMID:24890778

  9. Correlation between protein secondary structure, backbone bond angles, and side-chain orientations

    NASA Astrophysics Data System (ADS)

    Lundgren, Martin; Niemi, Antti J.

    2012-08-01

    We investigate the fine structure of the sp3 hybridized covalent bond geometry that governs the tetrahedral architecture around the central Cα carbon of a protein backbone, and for this we develop new visualization techniques to analyze high-resolution x-ray structures in the Protein Data Bank. We observe that there is a correlation between the deformations of the ideal tetrahedral symmetry and the local secondary structure of the protein. We propose a universal coarse-grained energy function to describe the ensuing side-chain geometry in terms of the Cβ carbon orientations. The energy function can model the side-chain geometry with a subatomic precision. As an example we construct the Cα-Cβ structure of HP35 chicken villin headpiece. We obtain a configuration that deviates less than 0.4 Å in root-mean-square distance from the experimental x-ray structure.

  10. Assignment of protein backbone resonances using connectivity, torsion angles and 13Calpha chemical shifts.

    PubMed

    Morris, Laura C; Valafar, Homayoun; Prestegard, James H

    2004-05-01

    A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just (13)C(alpha) chemical shifts (delta((13)C(alpha))) and data restricting the phi and psi backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in delta((13)C(alpha)), phi, and psi space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and (13)C(alpha) chemical shift data are available. PMID:15017135

  11. Collapse transition in proteins.

    PubMed

    Ziv, Guy; Thirumalai, D; Haran, Gilad

    2009-01-01

    The coil-globule transition, a tenet of the physics of polymers, has been identified in recent years as an important unresolved aspect of the initial stages of the folding of proteins. We describe the basics of the collapse transition, starting with homopolymers and continuing with proteins. Studies of denatured-state collapse under equilibrium are then presented. An emphasis is placed on single-molecule fluorescence experiments, which are particularly useful for measuring properties of the denatured state even under conditions of coexistence with the folded state. Attempts to understand the dynamics of collapse, both theoretically and experimentally, are then described. Only an upper limit for the rate of collapse has been obtained so far. Improvements in experimental and theoretical methodology are likely to continue to push our understanding of the importance of the denatured-state thermodynamics and dynamics for protein folding in the coming years. PMID:19081910

  12. Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

    SciTech Connect

    Naritomi, Yusuke; Fuchigami, Sotaro

    2013-12-07

    We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of C{sub α} atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

  13. Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

    NASA Astrophysics Data System (ADS)

    Naritomi, Yusuke; Fuchigami, Sotaro

    2013-12-01

    We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of Cα atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

  14. Water proton spin saturation affects measured protein backbone 15 N spin relaxation rates

    NASA Astrophysics Data System (ADS)

    Chen, Kang; Tjandra, Nico

    2011-12-01

    Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T1 and transversal rotating-frame T1ρ spin relaxation. Unexpectedly the 15N T1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichiacoli. glutamine binding protein (GlnBP) the T1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T1ρ data sets are correlated without an apparent offset. The average T1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios ( s/ n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1H N recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (< S2> = 0.81) than the non-saturated ones (< S2> = 0.88), indicating such order parameters may be previously underestimated.

  15. Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein.

    PubMed Central

    Gao, Y.; Kaluarachchi, K.; Giedroc, D. P.

    1998-01-01

    Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core

  16. A Multi-Objective Approach for Protein Structure Prediction Based on an Energy Model and Backbone Angle Preferences

    PubMed Central

    Tsay, Jyh-Jong; Su, Shih-Chieh; Yu, Chin-Sheng

    2015-01-01

    Protein structure prediction (PSP) is concerned with the prediction of protein tertiary structure from primary structure and is a challenging calculation problem. After decades of research effort, numerous solutions have been proposed for optimisation methods based on energy models. However, further investigation and improvement is still needed to increase the accuracy and similarity of structures. This study presents a novel backbone angle preference factor, which is one of the factors inducing protein folding. The proposed multiobjective optimisation approach simultaneously considers energy models and backbone angle preferences to solve the ab initio PSP. To prove the effectiveness of the multiobjective optimisation approach based on the energy models and backbone angle preferences, 75 amino acid sequences with lengths ranging from 22 to 88 amino acids were selected from the CB513 data set to be the benchmarks. The data sets were highly dissimilar, therefore indicating that they are meaningful. The experimental results showed that the root-mean-square deviation (RMSD) of the multiobjective optimization approach based on energy model and backbone angle preferences was superior to those of typical energy models, indicating that the proposed approach can facilitate the ab initio PSP. PMID:26151847

  17. Unconventional N-H…N Hydrogen Bonds Involving Proline Backbone Nitrogen in Protein Structures.

    PubMed

    Deepak, R N V Krishna; Sankararamakrishnan, Ramasubbu

    2016-05-10

    Contrary to DNA double-helical structures, hydrogen bonds (H-bonds) involving nitrogen as the acceptor are not common in protein structures. We systematically searched N-H…N H-bonds in two different sets of protein structures. Data set I consists of neutron diffraction and ultrahigh-resolution x-ray structures (0.9 Å resolution or better) and the hydrogen atom positions in these structures were determined experimentally. Data set II contains structures determined using x-ray diffraction (resolution ≤ 1.8 Å) and the positions of hydrogen atoms were generated using a computational method. We identified 114 and 14,347 potential N-H…N H-bonds from these two data sets, respectively, and 56-66% of these were of the Ni+1-Hi+1…Ni type, with Ni being the proline backbone nitrogen. To further understand the nature of such unusual contacts, we performed quantum chemical calculations on the model compound N-acetyl-L-proline-N-methylamide (Ace-Pro-NMe) with coordinates taken from the experimentally determined structures. A potential energy profile generated by varying the ψ dihedral angle in Ace-Pro-NMe indicates that the conformation with the N-H…N H-bond is the most stable. An analysis of H-bond-forming proline residues reveals that more than 30% of the proline carbonyl groups are also involved in n → π(∗) interactions with the carbonyl carbon of the preceding residue. Natural bond orbital analyses demonstrate that the strength of N-H…N H-bonds is less than half of that observed for a conventional H-bond. This study clearly establishes the H-bonding capability of proline nitrogen and its prevalence in protein structures. We found many proteins with multiple instances of H-bond-forming prolines. With more than 15% of all proline residues participating in N-H…N H-bonds, we suggest a new, to our knowledge, structural role for proline in providing stability to loops and capping regions of secondary structures in proteins. PMID:27166805

  18. Protein inhibitors of serine proteinases: role of backbone structure and dynamics in controlling the hydrolysis constant.

    PubMed

    Song, Jikui; Markley, John L

    2003-05-13

    Standard mechanism protein inhibitors of serine proteinases bind as substrates and are cleaved by cognate proteinases at their reactive sites. The hydrolysis constant for this cleavage reaction at the P(1)-P(1)' peptide bond (K(hyd)) is determined by the relative concentrations at equilibrium of the "intact" (uncleaved, I) and "modified" (reactive site cleaved, I*) forms of the inhibitor. The pH dependence of K(hyd) can be explained in terms of a pH-independent term, K(hyd) degrees, plus the proton dissociation constants of the newly formed amino and carboxylate groups at the cleavage site. Two protein inhibitors that differ from one another by a single residue substitution have been found to have K(hyd) degrees values that differ by a factor of 5 [Ardelt, W., and Laskowski, M., Jr. (1991) J. Mol. Biol. 220, 1041-1052]: turkey ovomucoid third domain (OMTKY3) has K(hyd) degrees = 1.0, and Indian peafowl ovomucoid third domain (OMIPF3), which differs from OMTKY3 by the substitution P(2)'-Tyr(20)His, has K(hyd) degrees = 5.15. What mechanism is responsible for this small difference? Is it structural (enthalpic) or dynamic (entropic)? Does the mutation affect the free energy of the I state, the I* state, or both? We have addressed these questions through NMR investigations of the I and I forms of OMTKY3 and OMIPF3. Information about structure was derived from measurements of NMR chemical shift changes and trans-hydrogen-bond J-couplings; information about dynamics was obtained through measurements of (15)N relaxation rates and (1)H-(15)N heteronuclear NOEs with model-free analysis of the results. Although the I forms of each variant are more dynamic than the corresponding I forms, the study revealed no appreciable difference in the backbone dynamics of either intact inhibitor (OMIPF3 vs OMTKY3) or modified inhibitor (OMIPF3* vs OMTKY3*). Instead, changes in chemical shifts and trans-hydrogen-bond J-couplings suggested that the K(hyd) degrees difference arises from

  19. Comparison of Backbone Modification in Protein β-Sheets by α→γ Residue Replacement and α-Residue Methylation

    PubMed Central

    Lengyel, George A.; Reinert, Zachary E.; Griffith, Brian D.

    2014-01-01

    The mimicry of protein tertiary structure by oligomers with unnatural backbones is a significant contemporary research challenge. Among common elements of secondary structure found in natural proteins, sheets have proven the most difficult to address. Here, we report the systematic comparison of different strategies for peptide backbone modification in β-sheets with the goal of identifying the best method for replacing a multi-stranded sheet in a protein tertiary fold. The most effective sheet modifications examined lead to native-like tertiary folding behavior with thermodynamic fold stability comparable to the prototype protein on which the modified backbones are based. PMID:24909436

  20. Reduced dimensionality (4,3)D-hnCOCANH experiment: an efficient backbone assignment tool for NMR studies of proteins.

    PubMed

    Kumar, Dinesh

    2013-09-01

    Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone ((1)H, (15)N, (13)C(α) and (13)C') resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment--(4,3)D-hnCOCANH and exploits the linear combinations of backbone ((13)C(α) and (13)C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks--which is different in sum (CA + CO) and difference (CA - CO) frequency regions--greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide (15)N and backbone (13)C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (i→i + 1) (15)N and (13)C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F₂-F₃ planes of the spectrum corresponding to sum (CA + CO) and difference (CA - CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW

  1. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning

    PubMed Central

    Heffernan, Rhys; Paliwal, Kuldip; Lyons, James; Dehzangi, Abdollah; Sharma, Alok; Wang, Jihua; Sattar, Abdul; Yang, Yuedong; Zhou, Yaoqi

    2015-01-01

    Direct prediction of protein structure from sequence is a challenging problem. An effective approach is to break it up into independent sub-problems. These sub-problems such as prediction of protein secondary structure can then be solved independently. In a previous study, we found that an iterative use of predicted secondary structure and backbone torsion angles can further improve secondary structure and torsion angle prediction. In this study, we expand the iterative features to include solvent accessible surface area and backbone angles and dihedrals based on Cα atoms. By using a deep learning neural network in three iterations, we achieved 82% accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone φ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively, for an independent test dataset of 1199 proteins. The accuracy of the method is slightly lower for 72 CASP 11 targets but much higher than those of model structures from current state-of-the-art techniques. This suggests the potentially beneficial use of these predicted properties for model assessment and ranking. PMID:26098304

  2. DNA Backbone BI/BII Distribution and Dynamics in E2 Protein-Bound Environment Determined by Molecular Dynamics Simulations.

    PubMed

    Robertson, James C; Cheatham, Thomas E

    2015-11-01

    BI and BII conformational substates in the DNA backbone typify canonical B-form DNA. The BI and BII substates are important for structural variation of DNA and have been implicated in protein-nucleic acid recognition mechanisms. Recent refinements have been made to nucleic acid force fields employed in molecular dynamics simulations that demonstrate a better ability to model the BI and BII states, leading to overall improved modeling of DNA structure and dynamics. These force field improvements have yet to be significantly demonstrated in the context of a protein-DNA system extended to long time scales. Our plan was to run molecular dynamics simulations of a well-studied protein-DNA system (E2-DNA) into the microsecond time scale and determine the ability of the force field to populate BII states in the DNA backbone consistent with dinucleotide steps crystallized in the BII conformation. The results showed that the dinucleotide steps in the E2-DNA complex with the highest BII populations from simulation trajectories corresponded to the dinucleotide steps crystallized in the BII state and that decoy BI and BII states converge to the same results within approximately one microsecond. PMID:26482568

  3. The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog

    PubMed Central

    Cuneo, Matthew J; Tian, Yaji; Allert, Malin; Hellinga, Homme W

    2008-01-01

    Background Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability. Results We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 Å resolution. We find that tteRBP is significantly more stable (appTm value ~102°C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56°C). The tteRBP has essentially the identical backbone conformation (0.41 Å RMSD of 235/271 Cα positions and 0.65 Å RMSD of 270/271 Cα positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities. Conclusion The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences. PMID:18373848

  4. General order parameter based correlation analysis of protein backbone motions between experimental NMR relaxation measurements and molecular dynamics simulations

    SciTech Connect

    Liu, Qing; Shi, Chaowei; Yu, Lu; Zhang, Longhua; Xiong, Ying; Tian, Changlin

    2015-02-13

    Internal backbone dynamic motions are essential for different protein functions and occur on a wide range of time scales, from femtoseconds to seconds. Molecular dynamic (MD) simulations and nuclear magnetic resonance (NMR) spin relaxation measurements are valuable tools to gain access to fast (nanosecond) internal motions. However, there exist few reports on correlation analysis between MD and NMR relaxation data. Here, backbone relaxation measurements of {sup 15}N-labeled SH3 (Src homology 3) domain proteins in aqueous buffer were used to generate general order parameters (S{sup 2}) using a model-free approach. Simultaneously, 80 ns MD simulations of SH3 domain proteins in a defined hydrated box at neutral pH were conducted and the general order parameters (S{sup 2}) were derived from the MD trajectory. Correlation analysis using the Gromos force field indicated that S{sup 2} values from NMR relaxation measurements and MD simulations were significantly different. MD simulations were performed on models with different charge states for three histidine residues, and with different water models, which were SPC (simple point charge) water model and SPC/E (extended simple point charge) water model. S{sup 2} parameters from MD simulations with charges for all three histidines and with the SPC/E water model correlated well with S{sup 2} calculated from the experimental NMR relaxation measurements, in a site-specific manner. - Highlights: • Correlation analysis between NMR relaxation measurements and MD simulations. • General order parameter (S{sup 2}) as common reference between the two methods. • Different protein dynamics with different Histidine charge states in neutral pH. • Different protein dynamics with different water models.

  5. General trends of dihedral conformational transitions in a globular protein.

    PubMed

    Miao, Yinglong; Baudry, Jerome; Smith, Jeremy C; McCammon, J Andrew

    2016-04-01

    Dihedral conformational transitions are analyzed systematically in a model globular protein, cytochrome P450cam, to examine their structural and chemical dependences through combined conventional molecular dynamics (cMD), accelerated molecular dynamics (aMD) and adaptive biasing force (ABF) simulations. The aMD simulations are performed at two acceleration levels, using dihedral and dual boost, respectively. In comparison with cMD, aMD samples protein dihedral transitions approximately two times faster on average using dihedral boost, and ∼ 3.5 times faster using dual boost. In the protein backbone, significantly higher dihedral transition rates are observed in the bend, coil, and turn flexible regions, followed by the β bridge and β sheet, and then the helices. Moreover, protein side chains of greater length exhibit higher transition rates on average in the aMD-enhanced sampling. Side chains of the same length (particularly Nχ = 2) exhibit decreasing transition rates with residues when going from hydrophobic to polar, then charged and aromatic chemical types. The reduction of dihedral transition rates is found to be correlated with increasing energy barriers as identified through ABF free energy calculations. These general trends of dihedral conformational transitions provide important insights into the hierarchical dynamics and complex free energy landscapes of functional proteins. PMID:26799251

  6. The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog

    SciTech Connect

    Cuneo, Matthew J.; Tian, Yaji; Allert, Malin; Hellinga, Homme W.

    2008-10-27

    We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 {angstrom} resolution. We find that tteRBP is significantly more stable ({sup app}T{sub m} value {approx} 102 C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) ({sup app}T{sub m} value {approx} 56 C). The tteRBP has essentially the identical backbone conformation (0.41 {angstrom} RMSD of 235/271 C{sub {alpha}} positions and 0.65 {angstrom} RMSD of 270/271 C{sub {alpha}} positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities.

  7. Backbone upgrades and DEC equipment replacement

    NASA Technical Reports Server (NTRS)

    Vancamp, Warren

    1991-01-01

    The NASA Science Internet (NSI) dual protocol backbone is outlined. It includes DECnet link upgrades to match TCP/IP link performance. It also includes the integration of backbone resources and central management. The phase 1 transition process is outlined.

  8. A HAUSDORFF-BASED NOE ASSIGNMENT ALGORITHM USING PROTEIN BACKBONE DETERMINED FROM RESIDUAL DIPOLAR COUPLINGS AND ROTAMER PATTERNS

    PubMed Central

    Zeng, Jianyang (Michael); Tripathy, Chittaranjan; Zhou, Pei; Donald, Bruce R.

    2008-01-01

    High-throughput structure determination based on solution Nuclear Magnetic Resonance (NMR) spectroscopy plays an important role in structural genomics. One of the main bottlenecks in NMR structure determination is the interpretation of NMR data to obtain a sufficient number of accurate distance restraints by assigning nuclear Overhauser effect (NOE) spectral peaks to pairs of protons. The difficulty in automated NOE assignment mainly lies in the ambiguities arising both from the resonance degeneracy of chemical shifts and from the uncertainty due to experimental errors in NOE peak positions. In this paper we present a novel NOE assignment algorithm, called HAusdorff-based NOE Assignment (HANA), that starts with a high-resolution protein backbone computed using only two residual dipolar couplings (RDCs) per residue37, 39, employs a Hausdorff-based pattern matching technique to deduce similarity between experimental and back-computed NOE spectra for each rotamer from a statistically diverse library, and drives the selection of optimal position-specific rotamers for filtering ambiguous NOE assignments. Our algorithm runs in time O(tn3 +tn log t), where t is the maximum number of rotamers per residue and n is the size of the protein. Application of our algorithm on biological NMR data for three proteins, namely, human ubiquitin, the zinc finger domain of the human DNA Y-polymerase Eta (pol η) and the human Set2-Rpb1 interacting domain (hSRI) demonstrates that our algorithm overcomes spectral noise to achieve more than 90% assignment accuracy. Additionally, the final structures calculated using our automated NOE assignments have backbone RMSD < 1.7 Å and all-heavy-atom RMSD < 2.5 Å from reference structures that were determined either by X-ray crystallography or traditional NMR approaches. These results show that our NOE assignment algorithm can be successfully applied to protein NMR spectra to obtain high-quality structures. PMID:19122773

  9. Insight into a molecular interaction force supporting peptide backbones and its implication to protein loops and folding

    PubMed Central

    Du, Qi-Shi; Chen, Dong; Xie, Neng-Zhong; Huang, Ri-Bo; Chou, Kuo-Chen

    2015-01-01

    Although not being classified as the most fundamental protein structural elements like α-helices and β-strands, the loop segment may play considerable roles for protein stability, flexibility, and dynamic activity. Meanwhile, the protein loop is also quite elusive; i.e. its interactions with the other parts of protein as well as its own shape-maintaining forces have still remained as a puzzle or at least not quite clear yet. Here, we report a molecular force, the so-called polar hydrogen–π interaction (Hp–π), which may play an important role in supporting the backbones of protein loops. By conducting the potential energy surface scanning calculations on the quasi π-plane of peptide bond unit, we have observed the following intriguing phenomena: (1) when the polar hydrogen atom of a peptide unit is perpendicularly pointing to the π-plane of other peptide bond units, a remarkable Hp–π interaction occurs; (2) the interaction is distance and orientation dependent, acting in a broad space, and belonging to the ‘point-to-plane’ one. The molecular force reported here may provide useful interaction concepts and insights into better understanding the loop’s unique stability and flexibility feature, as well as the driving force of the protein global folding. PMID:25375237

  10. Insight into a molecular interaction force supporting peptide backbones and its implication to protein loops and folding.

    PubMed

    Du, Qi-Shi; Chen, Dong; Xie, Neng-Zhong; Huang, Ri-Bo; Chou, Kuo-Chen

    2015-09-01

    Although not being classified as the most fundamental protein structural elements like α-helices and β-strands, the loop segment may play considerable roles for protein stability, flexibility, and dynamic activity. Meanwhile, the protein loop is also quite elusive; i.e. its interactions with the other parts of protein as well as its own shape-maintaining forces have still remained as a puzzle or at least not quite clear yet. Here, we report a molecular force, the so-called polar hydrogen-π interaction (Hp-π), which may play an important role in supporting the backbones of protein loops. By conducting the potential energy surface scanning calculations on the quasi π-plane of peptide bond unit, we have observed the following intriguing phenomena: (1) when the polar hydrogen atom of a peptide unit is perpendicularly pointing to the π-plane of other peptide bond units, a remarkable Hp-π interaction occurs; (2) the interaction is distance and orientation dependent, acting in a broad space, and belonging to the 'point-to-plane' one. The molecular force reported here may provide useful interaction concepts and insights into better understanding the loop's unique stability and flexibility feature, as well as the driving force of the protein global folding. PMID:25375237

  11. Toward quantification of protein backbone–backbone hydrogen bonding energies: An energetic analysis of an amide-to-ester mutation in an α-helix within a protein

    PubMed Central

    Gao, Jianmin; Kelly, Jeffery W.

    2008-01-01

    Amide-to-ester backbone mutagenesis enables a specific backbone–backbone hydrogen bond (H-bond) in a protein to be eliminated in order to quantify its energetic contribution to protein folding. To extract a H-bonding free energy from an amide-to-ester perturbation free energy (ΔG folding,wt − ΔG folding,mut), it is necessary to correct for the putative introduction of a lone pair–lone pair electrostatic repulsion, as well as for the transfer free energy differences that may arise between the all amide sequence and the predominantly amide sequence harboring an ester bond. Mutation of the 9–10 amide bond within the V9F variant of the predominantly helical villin headpiece subdomain (HP35) to an ester or an E-olefin backbone bond results in a less stable but defined wild-type fold, an attribute required for this study. Comparing the folding free energies of the ester and E-olefin mutants, with correction for the desolvation free energy differences (ester and E-olefin) and the loss of an n-to-π* interaction (E-olefin), yields an experimentally based estimate of +0.4 kcal/mol for the O–O repulsion energy in an α-helical context, analogous to our previous experimentally based estimate of the O–O repulsion free energy in the context of a β-sheet. The small O–O repulsion energy indicates that amide-to-ester perturbation free energies can largely be attributed to the deletion of the backbone H-bonds after correction for desolvation differences. Quantitative evaluation of H-bonding in an α-helix should now be possible, an important step toward deciphering the balance of forces that enable spontaneous protein folding. PMID:18434500

  12. A Network Flow Approach to Predict Protein Targets and Flavonoid Backbones to Treat Respiratory Syncytial Virus Infection

    PubMed Central

    Poloni, Joice de Faria; Saraiva Macedo Timmers, Luis Fernando; Bonatto, Diego; Condessa Pitrez, Paulo Márcio; Tetelbom Stein, Renato

    2015-01-01

    Background. Respiratory syncytial virus (RSV) infection is the major cause of respiratory disease in lower respiratory tract in infants and young children. Attempts to develop effective vaccines or pharmacological treatments to inhibit RSV infection without undesired effects on human health have been unsuccessful. However, RSV infection has been reported to be affected by flavonoids. The mechanisms underlying viral inhibition induced by these compounds are largely unknown, making the development of new drugs difficult. Methods. To understand the mechanisms induced by flavonoids to inhibit RSV infection, a systems pharmacology-based study was performed using microarray data from primary culture of human bronchial cells infected by RSV, together with compound-proteomic interaction data available for Homo sapiens. Results. After an initial evaluation of 26 flavonoids, 5 compounds (resveratrol, quercetin, myricetin, apigenin, and tricetin) were identified through topological analysis of a major chemical-protein (CP) and protein-protein interacting (PPI) network. In a nonclustered form, these flavonoids regulate directly the activity of two protein bottlenecks involved in inflammation and apoptosis. Conclusions. Our findings may potentially help uncovering mechanisms of action of early RSV infection and provide chemical backbones and their protein targets in the difficult quest to develop new effective drugs. PMID:25879022

  13. Isolation of the protein backbone of an arabinogalactan-protein from the styles of Nicotiana alata and characterization of a corresponding cDNA.

    PubMed Central

    Du, H; Simpson, R J; Moritz, R L; Clarke, A E; Bacic, A

    1994-01-01

    Arabinogalactan-proteins (AGPs) from the styles of Nicotiana alata were isolated by ion exchange and gel filtration chromatography. After deglycosylation by anhydrous hydrogen fluoride, the protein backbones were fractionated by reversed-phase HPLC. One of the protein backbones, containing mainly hydroxyproline, alanine, and serine residues (53% of total residues), was digested with proteases, and the peptides were isolated and sequenced. This sequence information allowed the cloning of a 712-bp cDNA, AGPNa1. AGPNa1 encodes a 132-amino acid protein with three domains: an N-terminal secretion signal sequence, which is cleaved from the mature protein; a central sequence, which contains most of the hydroxyproline/proline residues; and a C-terminal hydrophobic region. AGPNa1 is expressed in many tissues of N. alata and related species. The arrangement of domains and amino acid composition of the AGP encoded by AGPNa1 are similar to that of an AGP from pear cell suspension culture filtrate, although the only sequence identity is at the N termini of the mature proteins. PMID:7827496

  14. Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks.

    PubMed

    de Brevern, A G; Etchebest, C; Hazout, S

    2000-11-15

    By using an unsupervised cluster analyzer, we have identified a local structural alphabet composed of 16 folding patterns of five consecutive C(alpha) ("protein blocks"). The dependence that exists between successive blocks is explicitly taken into account. A Bayesian approach based on the relation protein block-amino acid propensity is used for prediction and leads to a success rate close to 35%. Sharing sequence windows associated with certain blocks into "sequence families" improves the prediction accuracy by 6%. This prediction accuracy exceeds 75% when keeping the first four predicted protein blocks at each site of the protein. In addition, two different strategies are proposed: the first one defines the number of protein blocks in each site needed for respecting a user-fixed prediction accuracy, and alternatively, the second one defines the different protein sites to be predicted with a user-fixed number of blocks and a chosen accuracy. This last strategy applied to the ubiquitin conjugating enzyme (alpha/beta protein) shows that 91% of the sites may be predicted with a prediction accuracy larger than 77% considering only three blocks per site. The prediction strategies proposed improve our knowledge about sequence-structure dependence and should be very useful in ab initio protein modelling. PMID:11025540

  15. Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

    PubMed

    Singh, Vijay; Wang, Shenliang; Kool, Eric T

    2013-04-24

    Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy. PMID:23590213

  16. Prediction of Mutational Tolerance in HIV-1 Protease and Reverse Transcriptase Using Flexible Backbone Protein Design

    PubMed Central

    Varela, Rocco; Ó Conchúir, Shane; Kortemme, Tanja

    2012-01-01

    Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model. PMID:22927804

  17. High Accuracy of Karplus Equations for Relating Three-Bond J Couplings to Protein Backbone Torsion Angles

    PubMed Central

    Li, Fang; Lee, Jung Ho; Grishaev, Alexander; Ying, Jinfa; Bax, Ad

    2015-01-01

    3JC′C′ and 3JHNHα couplings are related to the intervening backbone torsion angle ϕ by standard Karplus equations. Although these couplings are known to be affected by parameters other than ϕ, including H-bonding, valence angles and residue type, experimental results and quantum calculations indicate that the impact of these latter parameters is typically very small. The solution NMR structure of protein GB3, newly refined by using extensive sets of residual dipolar couplings (RDCs), yields 50–60% better Karplus equation agreement between ϕ angles and experimental 3JC′C′ and 3JHNHα values than does the high resolution X-ray structure. In intrinsically disordered proteins, 3JC′C′ and 3JHNHα couplings can be measured at even higher accuracy, and the impact of factors other than the intervening torsion angle on 3J will be smaller than in folded proteins, making these couplings exceptionally valuable reporters on the ensemble of ϕ angles sampled by each residue. PMID:25511552

  18. Independent Metrics for Protein Backbone and Side-Chain Flexibility: Time Scales and Effects of Ligand Binding.

    PubMed

    Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R

    2015-03-10

    Conformational dynamics are central for understanding biomolecular structure and function, since biological macromolecules are inherently flexible at room temperature and in solution. Computational methods are nowadays capable of providing valuable information on the conformational ensembles of biomolecules. However, analysis tools and intuitive metrics that capture dynamic information from in silico generated structural ensembles are limited. In standard work-flows, flexibility in a conformational ensemble is represented through residue-wise root-mean-square fluctuations or B-factors following a global alignment. Consequently, these approaches relying on global alignments discard valuable information on local dynamics. Results inherently depend on global flexibility, residue size, and connectivity. In this study we present a novel approach for capturing positional fluctuations based on multiple local alignments instead of one single global alignment. The method captures local dynamics within a structural ensemble independent of residue type by splitting individual local and global degrees of freedom of protein backbone and side-chains. Dependence on residue type and size in the side-chains is removed via normalization with the B-factors of the isolated residue. As a test case, we demonstrate its application to a molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) on the millisecond time scale. This allows for illustrating different time scales of backbone and side-chain flexibility. Additionally, we demonstrate the effects of ligand binding on side-chain flexibility of three serine proteases. We expect our new methodology for quantifying local flexibility to be helpful in unraveling local changes in biomolecular dynamics. PMID:26579739

  19. HMM-based prediction for protein structural motifs' two local properties: solvent accessibility and backbone torsion angles.

    PubMed

    Yu, Jianyong; Xiang, Leijun; Hong, Jiang; Zhang, Weidong

    2013-02-01

    Protein structure prediction is often assisted by predicting one-dimensional structural properties including relative solvent accessibility (RSA) surface and backbone torsion angles (BTA) of residues, and these two properties are continuously varying variables because proteins can move freely in a three-dimensional space. Instead of subdividing them into a few arbitrarily defined states that many popular approaches used, this paper proposes an integrated system for realvalue prediction of protein structural motifs' two local properties, based on the modified Hidden Markov Model that we previously presented. The model was used to capture the relevance of RSA and the dependency of BTA between adjacent residues along the local protein chain in motifs with definite probabilities. These two properties were predicted according to their own probability distribution. The method was applied to a protein fragment library. For nine different classes of motifs, real values of RSA were predicted with mean absolute error (MAE) of 0.122-0.175 and Pearson's correlation coefficient (PCC) of 0.623-0.714 between predicted and actual RSA. Meanwhile, real values of BTA were obtained with MAE of 8.5⁰-29.4⁰ for Φ angles, 11.2⁰-38.5⁰ for ψ angles and PCC of 0.601-0.716 for Φ, 0.597-0.713 for ψ. The results were compared with well-known Real-SPINE Server, and indicate the proposed method may at least serve as the foundation to obtain better local properties from structural motifs for protein structure prediction. PMID:22894152

  20. (4,2)D Projection--reconstruction experiments for protein backbone assignment: application to human carbonic anhydrase II and calbindin D(28K).

    PubMed

    Venters, Ronald A; Coggins, Brian E; Kojetin, Doug; Cavanagh, John; Zhou, Pei

    2005-06-22

    Projection-reconstruction NMR experiments have been shown to significantly reduce the acquisition time required to obtain protein backbone assignment data. To date, this concept has only been applied to smaller (15)N/(13)C-labeled proteins. Here, we show that projection-reconstruction NMR techniques can be extended to larger protonated and perdeuterated proteins. We present a suite of (4,2)D triple-resonance experiments for protein backbone assignment and a Hybrid Backprojection/Lower-Value algorithm for reconstructing data with relatively weak signal-to-noise ratios. In addition, we propose a sampling theorem and discuss its implication on the choice of projection angles. We demonstrate the efficacy of this approach using the 29 kDa protein, human carbonic anhydrase II and the 30 kDa protein, calbindin D(28K). PMID:15954785

  1. Assessing protein conformational sampling methods based on bivariate lag-distributions of backbone angles

    PubMed Central

    Maadooliat, Mehdi; Huang, Jianhua Z.

    2013-01-01

    Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence–structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu.edu/∼madoliat/LagSVD) that can be used to produce informative animations. PMID:22926831

  2. Synthesis and in vitro antioxidant functions of protein hydrolysate from backbones of Rastrelliger kanagurta by proteolytic enzymes.

    PubMed

    Sheriff, Sheik Abdulazeez; Sundaram, Balasubramanian; Ramamoorthy, Baranitharan; Ponnusamy, Ponmurugan

    2014-01-01

    Every year, a huge quantity of fishery wastes and by-products are generated by fish processing industries. These wastes are either underutilized to produce low market value products or dumped leading to environmental issues. Complete utilization of fishery wastes for recovering value added products would be beneficial to the society and individual. The fish protein hydrolysates and derived peptides of fishery resources are widely used as nutritional supplements, functional ingredients, and flavor enhancers in food, beverage and pharmaceutical industries. Antioxidants from fishery resources have attracted the attention of researchers as they are cheaper in cost, easy to derive, and do not have side effects. Thus the present investigation was designed to produce protein hydrolysate by pepsin and papain digestion from the backbones of Rastrelliger kanagurta (Indian mackerel) and evaluate its antioxidant properties through various in vitro assays. The results reveal that both hydrolysates are potent antioxidants, capable of scavenging 46% and 36% of DPPH (1,1-diphenyl-2 picrylhydrazyl) and 58.5% and 37.54% of superoxide radicals respectively. The hydrolysates exhibit significant (p < 0.05) reducing power and lipid peroxidation inhibition. Among the two hydrolysates produced, pepsin derived fraction is superior than papain derived fraction in terms of yield, DH (Degree of hydrolysis), and antioxidant activity. PMID:24596496

  3. Synthesis and in vitro antioxidant functions of protein hydrolysate from backbones of Rastrelliger kanagurta by proteolytic enzymes

    PubMed Central

    Sheriff, Sheik Abdulazeez; Sundaram, Balasubramanian; Ramamoorthy, Baranitharan; Ponnusamy, Ponmurugan

    2013-01-01

    Every year, a huge quantity of fishery wastes and by-products are generated by fish processing industries. These wastes are either underutilized to produce low market value products or dumped leading to environmental issues. Complete utilization of fishery wastes for recovering value added products would be beneficial to the society and individual. The fish protein hydrolysates and derived peptides of fishery resources are widely used as nutritional supplements, functional ingredients, and flavor enhancers in food, beverage and pharmaceutical industries. Antioxidants from fishery resources have attracted the attention of researchers as they are cheaper in cost, easy to derive, and do not have side effects. Thus the present investigation was designed to produce protein hydrolysate by pepsin and papain digestion from the backbones of Rastrelliger kanagurta (Indian mackerel) and evaluate its antioxidant properties through various in vitro assays. The results reveal that both hydrolysates are potent antioxidants, capable of scavenging 46% and 36% of DPPH (1,1-diphenyl-2 picrylhydrazyl) and 58.5% and 37.54% of superoxide radicals respectively. The hydrolysates exhibit significant (p < 0.05) reducing power and lipid peroxidation inhibition. Among the two hydrolysates produced, pepsin derived fraction is superior than papain derived fraction in terms of yield, DH (Degree of hydrolysis), and antioxidant activity. PMID:24596496

  4. Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

    PubMed

    Thomas, N E; Bramson, H N; Miller, W T; Kaiser, E T

    1987-07-14

    As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3663600

  5. Amplitudes of protein backbone dynamics and correlated motions in a small alpha/beta protein: correspondence of dipolar coupling and heteronuclear relaxation measurements.

    PubMed

    Clore, G Marius; Schwieters, Charles D

    2004-08-24

    Backbone residual dipolar coupling (N-H, Calpha-Halpha, N-C', and Calpha-C') data collected in five different media on the B3 IgG binding domain of streptococcal protein G (GB3) have been analyzed by simultaneous refinement of the coordinates and optimization of the magnitudes and orientations of the alignment tensors using single and multiple structure representations. We show, using appropriate error analysis, that agreement between observed and calculated dipolar couplings at the level of experimental uncertainty is obtained with a two-structure (N(e) = 2) ensemble representation which represents the simplest equilibrium description of anisotropic motions. The data permit one to determine the magnitude of the anisotropic motions along the four different backbone bond vectors in terms of order parameters. The order parameters, , for the N-H bond vectors are in qualitative agreement with the generalized order parameters, S(2)NH(relaxation), derived from (15)N relaxation measurements, with a correlation coefficient of 0.84. S(2)NH(relaxation) can be regarded as the product of an anisotropic order parameter, corresponding to derived from the residual dipolar couplings, and an axially symmetric order parameter, S(2)NH(axial), corresponding to bond librations which are expected to be essentially uniform along the polypeptide chain. The current data indicate that the average value of S(2)NH(axial) is approximately 0.9. The close correspondence of and S(2)NH(relaxation) indicates that any large-scale displacements from the mean coordinate positions on time scales longer than the rotational correlation time are rare and hence do not perturb the observed dipolar couplings. Analysis of a set of 100 N(e) = 2 ensembles reveals the presence of some long-range correlated motions of N-H and Calpha-Halpha vectors involving residues far apart in the sequence but close together in space. In addition, direct evidence is

  6. Drug screening strategy for human membrane proteins: from NMR protein backbone structure to in silica- and NMR-screened hits.

    PubMed

    Lindert, Steffen; Maslennikov, Innokentiy; Chiu, Ellis J C; Pierce, Levi C; McCammon, J Andrew; Choe, Senyon

    2014-03-21

    About 8000 genes encode membrane proteins in the human genome. The information about their druggability will be very useful to facilitate drug discovery and development. The main problem, however, consists of limited structural and functional information about these proteins because they are difficult to produce biochemically and to study. In this paper we describe the strategy that combines Cell-free protein expression, NMR spectroscopy, and molecular DYnamics simulation (CNDY) techniques. Results of a pilot CNDY experiment provide us with a guiding light towards expedited identification of the hit compounds against a new uncharacterized membrane protein as a potentially druggable target. These hits can then be further characterized and optimized to develop the initial lead compound quicker. We illustrate such "omics" approach for drug discovery with the CNDY strategy applied to two example proteins: hypoxia-induced genes HIGD1A and HIGD1B. PMID:24525125

  7. Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis.

    PubMed

    Zhou, Dan; Xiang, Lina; Zeng, Rongju; Cao, Fuhu; Zhu, Xiaoxi; Wang, Yanmei

    2011-12-01

    To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis. PMID:22038787

  8. A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein, an intrinsically disordered protein

    PubMed Central

    Mantsyzov, Alexey B; Maltsev, Alexander S; Ying, Jinfa; Shen, Yang; Hummer, Gerhard; Bax, Ad

    2014-01-01

    α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential HN–Hα and HN–HN NOEs, values for 3JHNHα, 1JHαCα, 2JCαN, and 1JCαN, as well as chemical shifts of 15N, 13Cα, and 13C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PPII region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the αR region (10–20%) is found. Analysis of 1H–1H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein. PMID:24976112

  9. Improving the prediction accuracy of residue solvent accessibility and real-value backbone torsion angles of proteins by guided-learning through a two-layer neural network

    PubMed Central

    Faraggi, Eshel; Xue, Bin; Zhou, Yaoqi

    2008-01-01

    This paper attempts to increase the prediction accuracy of residue solvent accessibility and real-value backbone torsion angles of proteins through improved learning. Most methods developed for improving the backpropagation algorithm of artificial neural networks are limited to small neural networks. Here, we introduce a guided-learning method suitable for networks of any size. The method employs a part of the weights for guiding and the other part for training and optimization. We demonstrate this technique by predicting residue solvent accessibility and real-value backbone torsion angles of proteins. In this application, the guiding factor is designed to satisfy the intuitive condition that for most residues, the contribution of a residue to the structural properties of another residue is smaller for greater separation in the protein-sequence distance between the two residues. We show that the guided-learning method makes a 2-4% reduction in ten-fold cross-validated mean absolute errors (MAE) for predicting residue solvent accessibility and backbone torsion angles, regardless of the size of database, the number of hidden layers and the size of input windows. This together with introduction of two-layer neural network with a bipolar activation function leads to a new method that has a MAE of 0.11 for residue solvent accessibility, 36° for ψ, and 22° for ϕ. The method is available as a Real-SPINE 3.0 server in http://sparks.informatics.iupui.edu. PMID:18704931

  10. "Pinning strategy": a novel approach for predicting the backbone structure in terms of protein blocks from sequence.

    PubMed

    De Brevern, A G; Etchebest, C; Benros, C; Hazout, S

    2007-01-01

    The description of protein 3D structures can be performed through a library of 3D fragments, named a structural alphabet. Our structural alphabet is composed of 16 small protein fragments of 5 C alpha in length, called protein blocks (PBs). It allows an efficient approximation of the 3D protein structures and a correct prediction of the local structure. The 72 most frequent series of 5 consecutive PBs, called structural words (SWs)are able to cover more than 90% of the 3D structures. PBs are highly conditioned by the presence of a limited number of transitions between them. In this study, we propose a new method called "pinning strategy" that used this specific feature to predict long protein fragments. Its goal is to define highly probable successions of PBs. It starts from the most probable SW and is then extended with overlapping SWs. Starting from an initial prediction rate of 34.4%, the use of the SWs instead of the PBs allows a gain of 4.5%. The pinning strategy simply applied to the SWs increases the prediction accuracy to 39.9%. In a second step, the sequence-structure relationship is optimized, the prediction accuracy reaches 43.6%. PMID:17426380

  11. On the source of entropic elastomeric force in polypeptides and proteins: Backbone configurational vs. side-chain solvational entropy

    SciTech Connect

    Luan, Chihao; Jaggard, J.; Harris, R.D.; Urry, D.W. )

    1989-01-01

    At physiological temperature in water, the relaxed state of the protein, elastin, and model elastomeric sequential polypeptides derived from this biological elastomer is the result of an inverse temperature transition. At lower temperatures, say at 20{degree}C, the hydrophobic side chains of the elastomers are hydrated with a low-entropy net of water. Following Flory and colleagues, thermoelasticity studies suggests that these polypeptide elastomers are dominantly entropic elastomers above the temperature of the inverse temperature transition. A central question becomes the source of the entropic elastomeric force. On stretching, hydrophobic side chains become exposed to water, resulting in an exothermic reaction of hydrophobic hydration. The issue addressed by the present report is whether this decrease in solvent entropy on stretching might make a major contribution to the entropic elastomeric restoring force. It has previously been argued that the free energy of solvation can be made very small by a 30% ethylene glycol (EG):70% water solvent mixture. This is demonstrated here using the repeating pentapeptide sequence of elastin (Val{sup 1}-Pro{sup 2}-Gly{sup 3} -Val{sup 4}-Gly{sup 5}){sub n} or poly(VPGVG) and its {gamma}-irradiation cross-linked elastomeric matrix. Differential scanning calorimetry of the inverse temperature transition of poly(VPGVG) shows the endothermic heat of the transition to become very small in EG/H{sub 2}O when compared with H{sub 2}O alone, which also indicates a very small entropy change for the transition on exposure of the hydrophobic side chains to the EG/H{sub 2}O solvent mixture. A similar result is found for the crosslinked elastomeric matrix. Significantly, however, in spite of the lower heats for hydrophobic solvation, the elastic modulus and the entropic elastomeric forces generated are greater in EG/H{sub 2}O.

  12. Grid-based backbone correction to the ff12SB protein force field for implicit-solvent simulations.

    PubMed

    Perez, Alberto; MacCallum, Justin L; Brini, Emiliano; Simmerling, Carlos; Dill, Ken A

    2015-10-13

    Force fields, such as Amber's ff12SB, can be fairly accurate models of the physical forces in proteins and other biomolecules. When coupled with accurate solvation models, force fields are able to bring insight into the conformational preferences, transitions, pathways, and free energies for these biomolecules. When computational speed/cost matters, implicit solvent is often used but at the cost of accuracy. We present an empirical grid-like correction term, in the spirit of cMAPs, to the combination of the ff12SB protein force field and the GBneck2 implicit-solvent model. Ff12SB-cMAP is parametrized on experimental helicity data. We provide validation on a set of peptides and proteins. Ff12SB-cMAP successfully improves the secondary structure biases observed in ff12SB + Gbneck2. Ff12SB-cMAP can be downloaded ( https://github.com/laufercenter/Amap.git ) and used within the Amber package. It can improve the agreement of force fields + implicit solvent with experiments. PMID:26574266

  13. A new approach to measuring protein backbone protection with high spatial resolution using H/D exchange and electron capture dissociation

    PubMed Central

    Abzalimov, Rinat R.; Bobst, Cedric E.; Kaltashov, Igor A.

    2013-01-01

    Inadequate spatial resolution remains one of the most serious limitations of hydrogen/deuterium exchange mass spectrometry (HDX MS), especially when applied to larger proteins (over 30 kDa). Supplementing proteolytic fragmentation of the protein in solution with ion dissociation in the gas phase has been used successfully by several groups to obtain near-residue level resolution. However, the restrictions imposed by the LC/MS/MS mode of operation on the data acquisition time frame makes it difficult in many cases to obtain signal-to-noise ratio adequate for reliable assignment of the backbone amide protection levels at individual residues. This restriction is lifted in the present work by eliminating the LC separation step from the workflow and taking advantage of the high resolving power and dynamic range of a Fourier transform ion cyclotron resonance mass spectrometer (FT ICR MS). A residue-level resolution is demonstrated for a peptic fragment of a 37 kDa recombinant protein (N-lobe of human serum transferrin) using electron-capture dissociation as an ion fragmentation tool. The absence of hydrogen scrambling in the gas phase prior to ion dissociation is verified using redundant HDX MS data generated by FT ICR MS. The backbone protection pattern generated by direct HDX MS/MS is in excellent agreement with the known crystal structure of the protein, but also provides information on conformational dynamics, which is not available from the static X-ray structure. PMID:23978257

  14. Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation.

    PubMed

    Vogel, Alexander; Reuther, Guido; Roark, Matthew B; Tan, Kui-Thong; Waldmann, Herbert; Feller, Scott E; Huster, Daniel

    2010-02-01

    The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data. PMID:19819220

  15. Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks

    NASA Astrophysics Data System (ADS)

    Kumar, Dinesh

    2013-12-01

    Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone 15N and 13C‧/13Cα chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone 15N(i) chemical shifts with that of 13C‧ (i - 1)/13Cα (i - 1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate 15N chemical shifts and (ii) reduces the signal overlap in F2(15N)-F3(1H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively.

  16. Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks.

    PubMed

    Kumar, Dinesh

    2013-12-01

    Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone (15)N and (13)C'/(13)C(α) chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone (15)N(i) chemical shifts with that of (13)C' (i-1)/(13)C(α) (i-1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate (15)N chemical shifts and (ii) reduces the signal overlap in F2((15)N)-F3((1)H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively. PMID:24161682

  17. Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins

    NASA Astrophysics Data System (ADS)

    Chevelkov, Veniamin; Habenstein, Birgit; Loquet, Antoine; Giller, Karin; Becker, Stefan; Lange, Adam

    2014-05-01

    Proton-detected solid-state NMR was applied to a highly deuterated insoluble, non-crystalline biological assembly, the Salmonella typhimurium type iii secretion system (T3SS) needle. Spectra of very high resolution and sensitivity were obtained at a low protonation level of 10-20% at exchangeable amide positions. We developed efficient experimental protocols for resonance assignment tailored for this system and the employed experimental conditions. Using exclusively dipolar-based interspin magnetization transfers, we recorded two sets of 3D spectra allowing for an almost complete backbone resonance assignment of the needle subunit PrgI. The additional information provided by the well-resolved proton dimension revealed the presence of two sets of resonances in the N-terminal helix of PrgI, while in previous studies employing 13C detection only a single set of resonances was observed.

  18. Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein

    SciTech Connect

    Yin, Cuifeng; Aramini, James M.; Ma, LiChung; Cort, John R.; Swapna, G.V.T.; Krug, R. M.; Montelione, Gaetano

    2011-10-01

    Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone 1HN, 15N, 13CO, and 13Ca, as well as side chain 13Cb, methyl (Ile-d1, Leu, Val), amide (Asn, Gln), and indole NH (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.

  19. Insight into the factors influencing the backbone dynamics of three homologous proteins, dendrotoxins I and K, and BPTI: FTIR and time-resolved fluorescence investigations.

    PubMed

    Hollecker, Michelle; Vincent, Michel; Gallay, Jacques; Ruysschaert, Jean-Marie; Goormaghtigh, Erik

    2002-12-24

    Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties. PMID:12484765

  20. RrgB321, a Fusion Protein of the Three Variants of the Pneumococcal Pilus Backbone RrgB, Is Protective In Vivo and Elicits Opsonic Antibodies

    PubMed Central

    Harfouche, Carole; Filippini, Sara; Gianfaldoni, Claudia; Ruggiero, Paolo; Moschioni, Monica; Maccari, Silvia; Pancotto, Laura; Arcidiacono, Letizia; Galletti, Bruno; Censini, Stefano; Mori, Elena; Giuliani, Marzia; Facciotti, Claudia; Cartocci, Elena; Savino, Silvana; Doro, Francesco; Pallaoro, Michele; Nocadello, Salvatore; Mancuso, Giuseppe; Haston, Mitch; Goldblatt, David; Barocchi, Michèle A.; Pizza, Mariagrazia; Rappuoli, Rino

    2012-01-01

    Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae. PMID:22083702

  1. Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

    PubMed

    Wilburn, Damien B; Bowen, Kathleen E; Doty, Kari A; Arumugam, Sengodagounder; Lane, Andrew N; Feldhoff, Pamela W; Feldhoff, Richard C

    2014-01-01

    In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP) superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF) is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s) without the immediate need for complementary mutations. Consequently, this unique

  2. Protein-Transition Metal Ion Networks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins obtained from agricultural sources were blended with divalent metal ions. Feather keratin, egg albumin, and wheat gluten showed increases of 2-3 times in modulus with addition of divalent transition metal ions Cu2+ and Zn2+. Increasing concentrations of ions resulted in increased stiffnes...

  3. Context and Force Field Dependence of the Loss of Protein Backbone Entropy upon Folding Using Realistic Denatured and Native State Ensembles

    PubMed Central

    Baxa, Michael C.; Haddadian, Esmael J.; Jha, Abhishek K.; Freed, Karl F.; Sosnick, Tobin R.

    2012-01-01

    The loss of conformational entropy is the largest unfavorable quantity affecting a protein’s stability. We calculate the reduction in the number of backbone conformations upon folding using the distribution of backbone dihedral angles (φ,ψ) obtained from an experimentally validated denatured state model, along with all-atom simulations for both the denatured and native states. The average loss of entropy per residue is TΔSBBU-N = 0.7, 0.9, or 1.1 kcal·mol−1 at T = 298 K, depending on the force field used, with a 0.6 kcal·mol−1 dispersion across the sequence. The average equates to a decrease of a factor of 3–7 in the number of conformations available per residue (f = ΩDenatured/ΩNative) or to a total of ftot=3n–7n for an n residue protein. Our value is smaller than most previous estimates where f = 7–20, i.e., our computed TΔSBBU-N is smaller by 10–100 kcal mol−1 for n=100. The differences emerge from our use of realistic native and denatured state ensembles as well as from the inclusion of accurate local sequence preferences, neighbor effects, and correlated motions (vibrations), in contrast to some previous studies that invoke gross assumptions about the entropy in either or both states. We find that the loss of entropy primarily depends on the local environment and less on properties of the native state, with the exception of α-helical residues in some force fields. PMID:22928488

  4. Backbone Relaxation Coupled to the Ionization of Internal Groups in Proteins: A Self-Guided Langevin Dynamics Study

    PubMed Central

    Damjanović, Ana; Wu, Xiongwu; García-Moreno E., Bertrand; Brooks, Bernard R.

    2008-01-01

    Pathways of structural relaxation triggered by ionization of internal groups in staphylococcal nuclease (SNase) were studied through multiple self-guided Langevin dynamics (SGLD) simulations. Circular dichroism, steady-state Trp fluorescence, and nuclear magnetic resonance spectroscopy have shown previously that variants of SNase with internal Glu, Asp, and Lys at positions 66 or 92, and Arg at position 66, exhibit local reorganization or global unfolding when the internal ionizable group is charged. Except for Arg-66, these internal ionizable groups have unusual pKa values and are neutral at physiological pH. The structural trends observed in the simulations are in general agreement with experimental observations. The I92D variant, which unfolds globally upon ionization of Asp-92, in simulations often exhibits extensive hydration of the protein core, and sometimes also significant perturbations of the β-barrel. In the crystal structure of the V66R variant, the β1 strand from the β-barrel is domain-swapped; in the simulations, the β1 strand is sometimes partially released. The V66K variant, which in solutions shows reorganization of six residues at the C-terminus of helix α1 and perturbations in the β-barrel structure, exhibits fraying of three residues of helix α1 in one simulation, and perturbations and partial unfolding of three β-strands in a few other simulations. In sharp contrast, very small structural changes were observed in simulations of the wild-type protein. The simulations indicate that charging of internal groups frequently triggers penetration of water into the protein interior. The pKa values of Asp-92 and Arg-66 calculated with continuum methods on SGLD-relaxed structures reached the normal values in most simulations. Detailed analysis of accuracy and performance of SGLD demonstrates that SGLD outperforms LD in sampling of alternative protein conformations without loss of the accuracy and level of detail characteristic of regular LD. PMID

  5. Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N–15N and carbonyl 13C–13C dipolar recoupling data

    PubMed Central

    Hu, Kan-Nian; Qiang, Wei; Bermejo, Guillermo A.; Schwieters, Charles D.; Tycko, Robert

    2013-01-01

    Recent structural studies of uniformly 15N, 13C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic 15N and 13C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole–dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole–dipole couplings among 15N-labeled backbone amide sites and among 13C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call 15N-BARE and 13C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. 15N-BARE and 13C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental 15N and 13C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the 15N-BARE and 13C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly 15N, 13C-labeled protein GB1. Results for GB1 show that 15N-BARE and 13C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures. PMID:22449573

  6. Strike a Balance: Optimization of Backbone Torsion Parameters of AMBER Polarizable Force Field for Simulations of Proteins and Peptides

    PubMed Central

    WANG, ZHI-XIANG; ZHANG, WEI; WU, CHUN; LEI, HONGXING; CIEPLAK, PIOTR; DUAN, YONG

    2014-01-01

    Based on the AMBER polarizable model (ff02), we have reoptimized the parameters related to the main-chain (Φ, Ψ) torsion angles by fitting to the Boltzmann-weighted average quantum mechanical (QM) energies of the important regions (i.e., β, PII, αR, and αL regions). Following the naming convention of the AMBER force field series, this release will be called ff02pol.rl The force field has been assessed both by energetic comparison against the QM data and by the replica exchange molecular dynamics simulations of short alanine peptides in water. For Ace-Ala-Nme, the simulated populations in the β, PII and αR regions were approximately 30, 43, and 26%, respectively. For Ace-(Ala)7-Nme, the populations in these three regions were approximately 24, 49, and 26%. Both were in qualitative agreement with the NMR and CD experimental conclusions. In comparison with the previous force field, ff02pol.rl demonstrated good balance among these three important regions. The optimized torsion parameters, together with those in ff02, allow us to carry out simulations on proteins and peptides with the consideration of polarization. PMID:16526038

  7. (1)H, (13)C and (15)N backbone and side-chain resonance assignment of the LAM-RRM1 N-terminal module of La protein from Dictyostelium discoideum.

    PubMed

    Chasapis, Christos T; Argyriou, Aikaterini I; Apostolidi, Maria; Konstantinidou, Parthena; Bentrop, Detlef; Stathopoulos, Constantinos; Spyroulias, Georgios A

    2015-10-01

    The N-terminal half of La protein consists of two concatenated motifs: La motif (LAM) and the N-terminal RNA recognition motif (RRM1) both of which are responsible for poly(U) RNA binding. Here, we present the backbone and side-chain assignments of the (1)H, (13)C and (15)N resonances of the 191-residue LAM-RRM1 region of the La protein from the lower eukaryote Dictyostelium discoideum and its secondary structure prediction. PMID:25687647

  8. The Na+-phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane insertion

    PubMed Central

    Kohl, Beate; Wagner, Carsten A; Huelseweh, Birgit; Busch, Andreas E; Werner, Andreas

    1998-01-01

    Renal handling of inorganic phosphate (Pi) involves a Na+-Pi cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi-II, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo. The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N- (1-3 and 1-5) and C-terminal (4-8 and 6-8) complementary fragments of NaPi-II from winter flounder. When the cognate fragments were used in combination (1-3 plus 4-8; 1-5 plus 6-8) they comprised the full complement of the putative transporter domains. None of the four individual fragments or the 1-5 plus 6-8 combination when expressed in Xenopus oocytes increased Pi flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaPi-II with regard to pH dependency and Km for Na+ and Pi binding; however, the maximal transport rate (vmax) was lower. Immunohistochemistry on cryosections confined the functionally active 1-3 plus 4-8 combination to the oocyte membrane. This was not the case for the 1-5 plus 6-8 combination or any of the individual fragments, all of which failed to induce fluorescence. A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N- nor C-terminal tags induced fluorescence. PMID:9508800

  9. Database algorithm for generating protein backbone and side-chain co-ordinates from a C alpha trace application to model building and detection of co-ordinate errors.

    PubMed

    Holm, L; Sander, C

    1991-03-01

    The problem of constructing all-atom model co-ordinates of a protein from an outline of the polypeptide chain is encountered in protein structure determination by crystallography or nuclear magnetic resonance spectroscopy, in model building by homology and in protein design. Here, we present an automatic procedure for generating full protein co-ordinates (backbone and, optionally, side-chains) given the C alpha trace and amino acid sequence. To construct backbones, a protein structure database is first scanned for fragments that locally fit the chain trace according to distance criteria. A best path algorithm then sifts through these segments and selects an optimal path with minimal mismatch at fragment joints. In blind tests, using fully known protein structures, backbones (C alpha, C, N, O) can be reconstructed with a reliability of 0.4 to 0.6 A root-mean-square position deviation and not more than 0 to 5% peptide flips. This accuracy is sufficient to identify possible errors in protein co-ordinate sets. To construct full co-ordinates, side-chains are added from a library of frequently occurring rotamers using a simple and fast Monte Carlo procedure with simulated annealing. In tests on X-ray structures determined at better than 2.5 A resolution, the positions of side-chain atoms in the protein core (less than 20% relative accessibility) have an accuracy of 1.6 A (r.m.s. deviation) and 70% of chi 1 angles are within 30 degrees of the X-ray structure. The computer program MaxSprout is available on request. PMID:2002501

  10. Motif-directed flexible backbone design of functional interactions

    PubMed Central

    Havranek, James J; Baker, David

    2009-01-01

    Computational protein design relies on a number of approximations to efficiently search the huge sequence space available to proteins. The fixed backbone and rotamer approximations in particular are important for formulating protein design as a discrete combinatorial optimization problem. However, the resulting coarse-grained sampling of possible side-chain terminal positions is problematic for the design of protein function, which depends on precise positioning of side-chain atoms. Although backbone flexibility can greatly increase the conformation freedom of side-chain functional groups, it is not obvious which backbone movements will generate the critical constellation of atoms responsible for protein function. Here, we report an automated method for identifying protein backbone movements that can give rise to any specified set of desired side-chain atomic placements and interactions, using protein–DNA interfaces as a model system. We use a library of previously observed protein–DNA interactions (motifs) and a rotamer-based description of side-chain conformation freedom to identify placements for the protein backbone that can give rise to a favorable side-chain interaction with DNA. We describe a tree-search algorithm for identifying those combinations of interactions from the library that can be realized with minimal perturbation of the protein backbone. We compare the efficiency of this method with the alternative approach of building and screening alternate backbone conformations. PMID:19472357

  11. Backbone 1H, 15N, and 13C resonance assignments and secondary structure of a novel protein OGL-20P(T)-358 from hyperthermophile Thermococcus thioreducens sp. nov.

    PubMed

    Wilson, Randall; Hughes, Ronny; Curto, Ernest; Ng, Joseph; Twigg, Pamela

    2007-12-31

    OGL-20P(T)-358 is a novel 66 amino acid residue protein from the hyperthermophile Thermococcus thioreducens sp. nov., strain OGL-20PT, which was collected from the wall of the hydrothermal black smoker in the Rainbow Vent along the mid-Atlantic ridge. This protein, which has no detectable sequence homology with proteins or domains of known function, has a calculated pI of 4.76 and a molecular mass of 8.2 kDa. We report here the backbone 1H, 15N, and 13C resonance assignments of OGL-20PT-358. Assignments are 97.5% (316/324) complete. Chemical shift index was used to determine the secondary structure of the protein, which appears to consist of primarily alpha-helical regions. This work is the foundation for future studies to determine the three-dimensional solution structure of the protein. PMID:18182861

  12. Effect of the chirality of the glycerol backbone on the bilayer and nonbilayer phase transitions in the diastereomers of di-dodecyl-beta-D-glucopyranosyl glycerol.

    PubMed Central

    Mannock, D A; Lewis, R N; McElhaney, R N; Akiyama, M; Yamada, H; Turner, D C; Gruner, S M

    1992-01-01

    We have studied the physical properties of aqueous dispersions of 1,2-sn- and 2,3-sn-didodecyl-beta-D-glucopyranosyl glycerols, as well as their diastereomeric mixture, using differential scanning calorimetry and low angle x-ray diffraction. Upon heating, both the chiral lipids and the diastereomeric mixture exhibit characteristically energetic L beta/L alpha phase transitions at 31.7-32.8 degrees C and two or three weakly energetic thermal events between 49 degrees C and 89 degrees C. In the diastereomeric mixture and the 1,2-sn glycerol derivative, these higher temperature endotherms correspond to the formation of, and interconversions between, several nonlamellar structures and have been assigned to L alpha/QIIa, QIIa/QIIb, and QIIb/HII phase transitions, respectively. The cubic phases QIIa and QIIb, whose cell lattice parameters are strongly temperature dependent, can be identified as belonging to space groups Ia3d and Pn3m/Pn3, respectively. In the equivalent 2,3-sn glucolipid, the QIIa phase is not observed and only two transitions are seen at 49 degrees C and 77 degrees C, which are identified as L alpha/QIIb and QIIb/HII phase transitions, respectively. These phase transitions temperatures are some 10 degrees C lower than those of the corresponding phase transitions observed in the diastereomeric mixture and the 1,2-sn glycerol derivative. On cooling, all three lipids exhibit a minor higher temperature exothermic event, which can be assigned to a HII/QIIb phase transition. An exothermic L alpha/L beta phase transition is observed at 30-31 degrees C. A shoulder is sometimes discernible on the high temperature side of the L alpha/L beta event, which may originate from a QIIb/L alpha phase transition prior to the freezing of the hydrocarbon chains. None of the lipids show evidence of a QIIa phase on cooling. No additional exothermic transitions are observed on further cooling to -3 degrees C. However, after nucleation at 0 degrees C followed by a short period

  13. Binding of transition metals to S100 proteins.

    PubMed

    Gilston, Benjamin A; Skaar, Eric P; Chazin, Walter J

    2016-08-01

    The S100 proteins are a unique class of EF-hand Ca(2+) binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn(2+), Cu(2+) and Mn(2+) ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function. PMID:27430886

  14. Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.

    PubMed

    Hefke, Frederik; Bagaria, Anurag; Reckel, Sina; Ullrich, Sandra Johanna; Dötsch, Volker; Glaubitz, Clemens; Güntert, Peter

    2011-02-01

    We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279 (1982)), types of amino acids are labeled with (13)C or/and (15)N such that cross peaks between (13)CO(i - 1) and (15)NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with (13)C and the second with (15)N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B(2)R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin. PMID:21170670

  15. Single molecule fluorescence experiments determine protein folding transition path times

    PubMed Central

    Chung, Hoi Sung; McHale, Kevin; Louis, John M.; Eaton, William A.

    2013-01-01

    The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs by crossing the free-energy barrier between two states. It is a single-molecule property that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding we determined the average transition-path time for a fast- and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule Förster-resonance-energy-transfer experiments. While the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by less than a factor of 5, showing that a fast-and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result. PMID:22363011

  16. Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity.

    PubMed

    Tremblay, Marie-Laurence; Xu, Lingling; Sarker, Muzaddid; Liu, Xiang-Qin; Rainey, Jan K

    2016-01-01

    Spider aciniform (wrapping) silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units). In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR) spectroscopy-based (15)N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps-ns timescale in the context of the single W unit (W₁) and the two unit concatemer (W₂). Unambiguous mapping of backbone dynamics throughout W₂ was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W₁ and W₂ reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre. PMID:27517921

  17. Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

    PubMed Central

    Tremblay, Marie-Laurence; Xu, Lingling; Sarker, Muzaddid; Liu, Xiang-Qin; Rainey, Jan K.

    2016-01-01

    Spider aciniform (wrapping) silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units). In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR) spectroscopy-based 15N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps–ns timescale in the context of the single W unit (W1) and the two unit concatemer (W2). Unambiguous mapping of backbone dynamics throughout W2 was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W1 and W2 reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre. PMID:27517921

  18. Parameterization of backbone flexibility in a coarse-grained force field for proteins (COFFDROP) derived from all-atom explicit-solvent molecular dynamics simulations of all possible two-residue peptides

    PubMed Central

    Frembgen-Kesner, Tamara; Andrews, Casey T.; Li, Shuxiang; Ngo, Nguyet Anh; Shubert, Scott A.; Jain, Aakash; Olayiwola, Oluwatoni; Weishaar, Mitch R.; Elcock, Adrian H.

    2015-01-01

    Recently, we reported the parameterization of a set of coarse-grained (CG) nonbonded potential functions, derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acid pairs, and designed for use in (implicit-solvent) Brownian dynamics (BD) simulations of proteins; this force field was named COFFDROP (COarse-grained Force Field for Dynamic Representations Of Proteins). Here, we describe the extension of COFFDROP to include bonded backbone terms derived from fitting to results of explicit-solvent MD simulations of all possible two-residue peptides containing the 20 standard amino acids, with histidine modeled in both its protonated and neutral forms. The iterative Boltzmann inversion (IBI) method was used to optimize new CG potential functions for backbone-related terms by attempting to reproduce angle, dihedral and distance probability distributions generated by the MD simulations. In a simple test of the transferability of the extended force field, the angle, dihedral and distance probability distributions obtained from BD simulations of 56 three-residue peptides were compared to results from corresponding explicit-solvent MD simulations. In a more challenging test of the COFFDROP force field, it was used to simulate eight intrinsically disordered proteins and was shown to quite accurately reproduce the experimental hydrodynamic radii (Rhydro), provided that the favorable nonbonded interactions of the force field were uniformly scaled downwards in magnitude. Overall, the results indicate that the COFFDROP force field is likely to find use in modeling the conformational behavior of intrinsically disordered proteins and multi-domain proteins connected by flexible linkers. PMID:26574429

  19. Instantaneous normal modes and the protein glass transition.

    PubMed

    Schulz, Roland; Krishnan, Marimuthu; Daidone, Isabella; Smith, Jeremy C

    2009-01-01

    In the instantaneous normal mode method, normal mode analysis is performed at instantaneous configurations of a condensed-phase system, leading to modes with negative eigenvalues. These negative modes provide a means of characterizing local anharmonicities of the potential energy surface. Here, we apply instantaneous normal mode to analyze temperature-dependent diffusive dynamics in molecular dynamics simulations of a small protein (a scorpion toxin). Those characteristics of the negative modes are determined that correlate with the dynamical (or glass) transition behavior of the protein, as manifested as an increase in the gradient with T of the average atomic mean-square displacement at approximately 220 K. The number of negative eigenvalues shows no transition with temperature. Further, although filtering the negative modes to retain only those with eigenvectors corresponding to double-well potentials does reveal a transition in the hydration water, again, no transition in the protein is seen. However, additional filtering of the protein double-well modes, so as to retain only those that, on energy minimization, escape to different regions of configurational space, finally leads to clear protein dynamical transition behavior. Partial minimization of instantaneous configurations is also found to remove nondiffusive imaginary modes. In summary, examination of the form of negative instantaneous normal modes is shown to furnish a physical picture of local diffusive dynamics accompanying the protein glass transition. PMID:19167298

  20. Instantaneous Normal Modes and the Protein Glass Transition

    SciTech Connect

    Schultz, Roland; Krishnan, Marimuthu; Daidone, Isabella; Smith, Jeremy C

    2009-01-01

    In the instantaneous normal mode method, normal mode analysis is performed at instantaneous configurations of a condensed-phase system, leading to modes with negative eigenvalues. These negative modes provide a means of characterizing local anharmonicities of the potential energy surface. Here, we apply instantaneous normal mode to analyze temperature-dependent diffusive dynamics in molecular dynamics simulations of a small protein (a scorpion toxin). Those characteristics of the negative modes are determined that correlate with the dynamical (or glass) transition behavior of the protein, as manifested as an increase in the gradient with T of the average atomic mean-square displacement at 220 K. The number of negative eigenvalues shows no transition with temperature. Further, although filtering the negative modes to retain only those with eigenvectors corresponding to double-well potentials does reveal a transition in the hydration water, again, no transition in the protein is seen. However, additional filtering of the protein double-well modes, so as to retain only those that, on energy minimization, escape to different regions of configurational space, finally leads to clear protein dynamical transition behavior. Partial minimization of instantaneous configurations is also found to remove nondiffusive imaginary modes. In summary, examination of the form of negative instantaneous normal modes is shown to furnish a physical picture of local diffusive dynamics accompanying the protein glass transition.

  1. Atomistic simulations of the MS2 coat protein conformational transition

    NASA Astrophysics Data System (ADS)

    Perkett, Matthew; Pontiggia, Francesco; Hagan, Michael

    2012-02-01

    During the replication of many viruses, hundreds to thousands of proteins self-assemble to form a protective protein coat, called a capsid, around the viral nucleic acid. Often these proteins have identical amino acid sequences with slightly different, or quasi-equivalent, conformations, which join in precise spatial arrangements. Although the structure of completed capsids is known to atomic resolution, little is known about the assembly intermediates and how protein conformations are selected during assembly. In this talk, we will use all-atom simulations to investigate how protein-RNA interactions guide conformational transitions of capsid proteins from the single-stranded RNA bacteriophage MS2. Since conformational changes occur on timescales which are not accessible to all-atom simulations, we use enhanced sampling methods to sample probable transition pathways and corresponding free energy profiles. Specifically, we will present free energy profiles associated with the MS2 capsid protein conformation in the presence and absence of RNA.

  2. Spectral and Hydration Dependence of Protein Dynamical Transition

    NASA Astrophysics Data System (ADS)

    Lipps, Ferdinand; Knab, J. R.; Chen, Jing Yin; He, Yunfen; Markelz, A. G.

    2008-03-01

    The protein dynamical transition, a rapid increase in flexibility at ˜ 200K, is hydration dependent suggesting that the transition may in fact be due to a transition in the surrounding water. Previously we have shown that the terahertz dielectric response is sensitive to the dynamical transition using terahertz time domain spectroscopy [1]. The broadband technique allows the determination of what motions are affected by the transition, that is whether long time scale motions such as side chain rotations, or faster vibrational motions. Here we examine both the frequency and hydration dependence of the protein dynamical transition for hydrated myoglobin powder for the 0.2 -- 2.0 THz and 80-295 K ranges. The transition is observed in both the real and imaginary parts of the dielectric response. Our earlier measurements of solutions did not show a transition in the real part of the permittivity, likely due to bulk solvent dominating the index. There is a strong frequency dependence with hydration. While a slight transition is observed at frequencies higher than 1 THz which is nearly hydration independent, for frequencies below 1 THz the strength of the transition rapidly increases with hydration. [1] A. G. Markelz, J. R. Knab, Jing Yin Chen, Yunfen He, Chem. Phys. Lett. 442, 413 (2007).

  3. Native proteins trap high-energy transit conformations

    PubMed Central

    Brereton, Andrew E.; Karplus, P. Andrew

    2015-01-01

    During protein folding and as part of some conformational changes that regulate protein function, the polypeptide chain must traverse high-energy barriers that separate the commonly adopted low-energy conformations. How distortions in peptide geometry allow these barrier-crossing transitions is a fundamental open question. One such important transition involves the movement of a non-glycine residue between the left side of the Ramachandran plot (that is, ϕ < 0°) and the right side (that is, ϕ > 0°). We report that high-energy conformations with ϕ ~ 0°, normally expected to occur only as fleeting transition states, are stably trapped in certain highly resolved native protein structures and that an analysis of these residues provides a detailed, experimentally derived map of the bond angle distortions taking place along the transition path. This unanticipated information lays to rest any uncertainty about whether such transitions are possible and how they occur, and in doing so lays a firm foundation for theoretical studies to better understand the transitions between basins that have been little studied but are integrally involved in protein folding and function. Also, the context of one such residue shows that even a designed highly stable protein can harbor substantial unfavorable interactions. PMID:26601321

  4. Protein Dynamical Transition: Role of Methyl Dynamics and Local Diffusion

    NASA Astrophysics Data System (ADS)

    Krishnan, M.; Schulz, R.; Smith, Jeremy C.

    2009-03-01

    The temperature-dependent protein dynamical transition is investigated using the Instanteous Normal mode analysis (INM) and molecular dynamics (MD) simulation of crystalline myoglobin and Toxin II. The onset of anharmonic dynamics in myoglobin is observed at 150 K, far below the much-studied solvent-activated dynamical transition at 220 K. A significant fraction of methyl groups exhibit nanosecond anharmonic rotational jump diffusion at 150 K indicating the essential role of methyl dynamics in the low-temperature onset of anharmonic protein dynamics. The methyl groups that exhibit many rotational excitations are located near xenon cavities, suggesting that cavities in proteins act as activation centers of anharmonic dynamics. INM analysis of Toxin II indicates the presence of non-zero barrier-crossing, diffusive degrees of freedom accessible to the protein below the dynamical transition. The number of these diffusive degrees of freedom increases abruptly at the dynamical transition. In summary, the present investigation suggests that local diffusive processes (for example, methyl dynamics) are activated at low temperatures (much below 220 K) leading to global diffusive protein dynamics (this involves excitation of many protein atoms) at the dynamical transition.

  5. Dynamical Transition of Protein-Hydration Water

    NASA Astrophysics Data System (ADS)

    Doster, W.; Busch, S.; Gaspar, A. M.; Appavou, M.-S.; Wuttke, J.; Scheer, H.

    2010-03-01

    Thin layers of water on biomolecular and other nanostructured surfaces can be supercooled to temperatures not accessible with bulk water. Chen et al. [Proc. Natl. Acad. Sci. U.S.A. 103, 9012 (2006)]PNASA60027-842410.1073/pnas.0602474103 suggested that anomalies near 220 K observed by quasielastic neutron scattering can be explained by a hidden critical point of bulk water. Based on more sensitive measurements of water on perdeuterated phycocyanin, using the new neutron backscattering spectrometer SPHERES, and an improved data analysis, we present results that show no sign of such a fragile-to-strong transition. The inflection of the elastic intensity at 220 K has a dynamic origin that is compatible with a calorimetric glass transition at 170 K. The temperature dependence of the relaxation times is highly sensitive to data evaluation; it can be brought into perfect agreement with the results of other techniques, without any anomaly.

  6. Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies

    SciTech Connect

    Ghosh, Arun K.; Leshchenko-Yashchuk, Sofiya; Anderson, David D.; Baldridge, Abigail; Noetzel, Marcus; Miller, Heather B.; Tie, Yunfeng; Wang, Yuan-Fang; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki

    2009-09-02

    Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1{prime}-ligands. These ligands are designed to interact with Gly-27{prime} carbonyl and Arg-8 side chain in the S1{prime}-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1{prime}-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1{prime} and S2 subsites of HIV-1 protease.

  7. Phase transitions in the assembly of multivalent signalling proteins

    SciTech Connect

    Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun; Kim, Soyeon; Chen, Baoyu; Guo, Liang; Llaguno, Marc; Hollingsworth, Javoris V.; King, David S.; Banani, Salman F.; Russo, Paul S.; Jiang, Qiu-Xing; Nixon, B. Tracy; Rosen, Michael K.

    2013-04-08

    Cells are organized on length scales ranging from angstrom to micrometers. However, the mechanisms by which angstrom-scale molecular properties are translated to micrometer-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometer-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

  8. Phase transitions in the assembly of multivalent signalling proteins.

    PubMed

    Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun; Kim, Soyeon; Chen, Baoyu; Guo, Liang; Llaguno, Marc; Hollingsworth, Javoris V; King, David S; Banani, Salman F; Russo, Paul S; Jiang, Qiu-Xing; Nixon, B Tracy; Rosen, Michael K

    2012-03-15

    Cells are organized on length scales ranging from ångström to micrometres. However, the mechanisms by which ångström-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology. PMID:22398450

  9. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins.

    PubMed

    Seki, Yohei; Ishiyama, Takashi; Sasaki, Daisuke; Abe, Junpei; Sohma, Youhei; Oisaki, Kounosuke; Kanai, Motomu

    2016-08-31

    Chemical modifications of native proteins can facilitate production of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, we report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallography. The absence of toxic metals and biochemically incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications. PMID:27534812

  10. Fluctuation Flooding Method (FFM) for accelerating conformational transitions of proteins

    NASA Astrophysics Data System (ADS)

    Harada, Ryuhei; Takano, Yu; Shigeta, Yasuteru

    2014-03-01

    A powerful conformational sampling method for accelerating structural transitions of proteins, "Fluctuation Flooding Method (FFM)," is proposed. In FFM, cycles of the following steps enhance the transitions: (i) extractions of largely fluctuating snapshots along anisotropic modes obtained from trajectories of multiple independent molecular dynamics (MD) simulations and (ii) conformational re-sampling of the snapshots via re-generations of initial velocities when re-starting MD simulations. In an application to bacteriophage T4 lysozyme, FFM successfully accelerated the open-closed transition with the 6 ns simulation starting solely from the open state, although the 1-μs canonical MD simulation failed to sample such a rare event.

  11. Simulation of Ames Backbone Network

    NASA Technical Reports Server (NTRS)

    Shahnasser, Hamid

    1998-01-01

    The networking demands of Ames Research Center are dramatically increasing. More and more workstations are requested to run video and audio applications on the network. These applications require a much greater bandwidth than data applications. The existing ARCLAN 2000 network bandwidth is insufficient, due to the use of FDDI as its backbone, for accommodating video applications. Operating at a maximum of 100 Mbps, FDDI can handle only a few workstations running multimedia applications. The ideal solution is to replace the current ARCLAN 2000 FDDI backbone with an ATM backbone. ATM has the capability to handle the increasing traffic loads on the ARCLAN 2000 that results from these new applications. As it can be seen from Figure 1, ARCLAN 2000 have a total of 32 routers (5 being core routers) each connected to the FDDI backbone via a 100 Mbps link. This network serves 34 different locations by using 34 hubs that are connected to secondary routers. End users are connected to the secondary routers with 10 Mbps links.

  12. NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone

    PubMed Central

    Awata, Junya; Takada, Saeko; Standley, Clive; Lechtreck, Karl F.; Bellvé, Karl D.; Pazour, Gregory J.; Fogarty, Kevin E.; Witman, George B.

    2014-01-01

    ABSTRACT The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. An nphp4-null mutant forms flagella with nearly normal length, ultrastructure and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but the amounts of certain membrane proteins were greatly reduced in the mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the transition zone barrier. PMID:25150219

  13. Structure-Based Prediction of Protein-Folding Transition Paths.

    PubMed

    Jacobs, William M; Shakhnovich, Eugene I

    2016-09-01

    We propose a general theory to describe the distribution of protein-folding transition paths. We show that transition paths follow a predictable sequence of high-free-energy transient states that are separated by free-energy barriers. Each transient state corresponds to the assembly of one or more discrete, cooperative units, which are determined directly from the native structure. We show that the transition state on a folding pathway is reached when a small number of critical contacts are formed between a specific set of substructures, after which folding proceeds downhill in free energy. This approach suggests a natural resolution for distinguishing parallel folding pathways and provides a simple means to predict the rate-limiting step in a folding reaction. Our theory identifies a common folding mechanism for proteins with diverse native structures and establishes general principles for the self-assembly of polymers with specific interactions. PMID:27602721

  14. Evaluation of Newcastle disease virus chimeras expressing the hemagglutinin-neuraminidase protein of velogenic strains in the context of a mesogenic recombinant virus backbone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major factor in the pathogenicity of Newcastle disease virus (NDV) is the amino acid sequence of the fusion protein cleavage site, but the role of other viral genes that contribute to virulence and different clinical forms of the disease remain undefined. To assess the role of other NDV genes in ...

  15. Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins

    PubMed Central

    Sanz-Hernández, Máximo; Vostrikov, Vitaly V.; Veglia, Gianluigi; De Simone, Alfonso

    2016-01-01

    The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that are often responsible for the biological function of oligomeric membrane protein states. PMID:26975211

  16. Adding Diverse Noncanonical Backbones to Rosetta: Enabling Peptidomimetic Design

    PubMed Central

    Craven, Timothy W.; Butterfoss, Glenn L.; Chou, Fang-Chieh; Lyskov, Sergey; Bullock, Brooke N.; Watkins, Andrew; Labonte, Jason W.; Pacella, Michael; Kilambi, Krishna Praneeth; Leaver-Fay, Andrew; Kuhlman, Brian; Gray, Jeffrey J.; Bradley, Philip; Kirshenbaum, Kent; Arora, Paramjit S.; Das, Rhiju; Bonneau, Richard

    2013-01-01

    Peptidomimetics are classes of molecules that mimic structural and functional attributes of polypeptides. Peptidomimetic oligomers can frequently be synthesized using efficient solid phase synthesis procedures similar to peptide synthesis. Conformationally ordered peptidomimetic oligomers are finding broad applications for molecular recognition and for inhibiting protein-protein interactions. One critical limitation is the limited set of design tools for identifying oligomer sequences that can adopt desired conformations. Here, we present expansions to the ROSETTA platform that enable structure prediction and design of five non-peptidic oligomer scaffolds (noncanonical backbones), oligooxopiperazines, oligo-peptoids, -peptides, hydrogen bond surrogate helices and oligosaccharides. This work is complementary to prior additions to model noncanonical protein side chains in ROSETTA. The main purpose of our manuscript is to give a detailed description to current and future developers of how each of these noncanonical backbones was implemented. Furthermore, we provide a general outline for implementation of new backbone types not discussed here. To illustrate the utility of this approach, we describe the first tests of the ROSETTA molecular mechanics energy function in the context of oligooxopiperazines, using quantum mechanical calculations as comparison points, scanning through backbone and side chain torsion angles for a model peptidomimetic. Finally, as an example of a novel design application, we describe the automated design of an oligooxopiperazine that inhibits the p53-MDM2 protein-protein interaction. For the general biological and bioengineering community, several noncanonical backbones have been incorporated into web applications that allow users to freely and rapidly test the presented protocols (http://rosie.rosettacommons.org). This work helps address the peptidomimetic community's need for an automated and expandable modeling tool for noncanonical

  17. ANSS Backbone Station Quality Assessment

    NASA Astrophysics Data System (ADS)

    Leeds, A.; McNamara, D.; Benz, H.; Gee, L.

    2006-12-01

    In this study we assess the ambient noise levels of the broadband seismic stations within the United States Geological Survey's (USGS) Advanced National Seismic System (ANSS) backbone network. The backbone consists of stations operated by the USGS as well as several regional network stations operated by universities. We also assess the improved detection capability of the network due to the installation of 13 additional backbone stations and the upgrade of 26 existing stations funded by the Earthscope initiative. This assessment makes use of probability density functions (PDF) of power spectral densities (PSD) (after McNamara and Buland, 2004) computed by a continuous noise monitoring system developed by the USGS- ANSS and the Incorporated Research Institutions in Seismology (IRIS) Data Management Center (DMC). We compute the median and mode of the PDF distribution and rank the stations relative to the Peterson Low noise model (LNM) (Peterson, 1993) for 11 different period bands. The power of the method lies in the fact that there is no need to screen the data for system transients, earthquakes or general data artifacts since they map into a background probability level. Previous studies have shown that most regional stations, instrumented with short period or extended short period instruments, have a higher noise level in all period bands while stations in the US network have lower noise levels at short periods (0.0625-8.0 seconds), high frequencies (8.0- 0.125Hz). The overall network is evaluated with respect to accomplishing the design goals set for the USArray/ANSS backbone project which were intended to increase broadband performance for the national monitoring network.

  18. 1H, 13C, 15N backbone and side chain NMR resonance assignments for the N-terminal RNA recognition motif of the HvGR-RBP1 protein involved in the regulation of barley (Hordeum vulgare L.) senescence

    PubMed Central

    Mason, Katelyn E.; Tripet, Brian P.; Parrott, David; Fischer, Andreas M.; Copié, Valérie

    2013-01-01

    Leaf senescence is an important process in the developmental life of all plant species. Senescence efficiency influences important agricultural traits such as grain protein content and plant growth, which are often limited by nitrogen use. Little is known about the molecular mechanisms regulating this highly orchestrated process. To enhance our understanding of leaf senescence and its regulation, we have undertaken the structural and functional characterization of previously unknown proteins that are involved in the control of senescence in barley (Hordeum vulgare L.). Previous microarray analysis highlighted several barley genes whose transcripts are differentially expressed during senescence, including a specific gene which is greater than 40 fold up-regulated in the flag leaves of early- as compared to late-senescing near-isogenic barley lines at 14 and 21 days past flowering (anthesis). From inspection of its amino acid sequence, this gene is predicted to encode a glycine-rich RNA-binding protein herein referred to as HvGR-RBP1. HvGR-RBP1 has been expressed as a recombinant protein in E. coli, and preliminary NMR data analysis has revealed that its glycine-rich C-terminal region [residues: 93–162] is structurally disordered whereas its N-terminal region [residues: 1–92] forms a well-folded domain. Herein, we report the complete 1H, 13C, and 15N resonance assignments of backbone and sidechain atoms, and the secondary structural topology of the N-terminal RNA Recognition Motif (RRM) domain of HvGR-RBP1, as a first step to unraveling its structural and functional role in the regulation of barley leaf senescence. PMID:23417794

  19. Physics-based potentials for the coupling between backbone- and side-chain-local conformational states in the united residue (UNRES) force field for protein simulations

    PubMed Central

    Sieradzan, Adam K.; Krupa, Paweł; Scheraga, Harold A.; Liwo, Adam; Czaplewski, Cezary

    2015-01-01

    The UNited RESidue (UNRES) model of polypeptide chains is a coarse-grained model in which each amino-acid residue is reduced to two interaction sites, namely a united peptide group (p) located halfway between the two neighboring α-carbon atoms (Cαs), which serve only as geometrical points, and a united side chain (SC) attached to the respective Cα. Owing to this simplification, millisecond Molecular Dynamics simulations of large systems can be performed. While UNRES predicts overall folds well, it reproduces the details of local chain conformation with lower accuracy. Recently, we implemented new knowledge-based torsional potentials (Krupa et. al. J. Chem. Theory Comput., 2013, 9, 4620–4632) that depend on the virtual-bond dihedral angles involving side chains: Cα ⋯ Cα ⋯ Cα ⋯ SC (τ(1)), SC ⋯ Cα ⋯ Cα ⋯ Cα (τ(2)), and SC ⋯ Cα ⋯ Cα ⋯ SC (τ(3)) in the UNRES force field. These potentials resulted in significant improvement of the simulated structures, especially in the loop regions. In this work, we introduce the physics-based counterparts of these potentials, which we derived from the all-atom energy surfaces of terminally-blocked amino-acid residues by Boltzmann integration over the angles λ(1) and λ(2) for rotation about the Cα ⋯ Cα virtual-bond angles and over the side-chain angles χ. The energy surfaces were, in turn, calculated by using the semiempirical AM1 method of molecular quantum mechanics. Entropy contribution was evaluated with use of the harmonic approximation from Hessian matrices. One-dimensional Fourier series in the respective virtual-bond-dihedral angles were fitted to the calculated potentials, and these expressions have been implemented in the UNRES force field. Basic calibration of the UNRES force field with the new potentials was carried out with eight training proteins, by selecting the optimal weight of the new energy terms and reducing the weight of the regular torsional terms. The force field was

  20. Conformation transition kinetics of Bombyx mori silk protein.

    PubMed

    Chen, Xin; Shao, Zhengzhong; Knight, David P; Vollrath, Fritz

    2007-07-01

    Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber. PMID:17436322

  1. Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R1ρ NMR Measurements: Application to Protein Backbone Dynamics Measurements.

    PubMed

    Kurauskas, Vilius; Weber, Emmanuelle; Hessel, Audrey; Ayala, Isabel; Marion, Dominique; Schanda, Paul

    2016-09-01

    Transverse relaxation rate measurements in magic-angle spinning solid-state nuclear magnetic resonance provide information about molecular motions occurring on nanosecond-to-millisecond (ns-ms) time scales. The measurement of heteronuclear ((13)C, (15)N) relaxation rate constants in the presence of a spin-lock radiofrequency field (R1ρ relaxation) provides access to such motions, and an increasing number of studies involving R1ρ relaxation in proteins have been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely, (1) the role of CSA/dipolar cross-correlated relaxation (CCR) and (2) the impact of fast proton spin flips (i.e., proton spin diffusion and relaxation). We show that CSA/D CCR in R1ρ experiments is measurable and that the CCR rate constant depends on ns-ms motions; it can thus provide insight into dynamics. We find that proton spin diffusion attenuates this CCR due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1ρ rate constants has practical advantages over the use of CCR rates, and this article reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation. PMID:27500976

  2. Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

    DOEpatents

    Kostic, N.M.; Chen, J.

    1991-03-05

    Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings

  3. Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

    DOEpatents

    Kostic, Nenad M.; Chen, Jian

    1991-03-05

    Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.

  4. Periodicity, planarity, and pixel (3P): a program using the intrinsic residual dipolar coupling periodicity-to-peptide plane correlation and phi/psi angles to derive protein backbone structures.

    PubMed

    Wang, Jinbu; Walsh, Joseph D; Kuszewski, John; Wang, Yun-Xing

    2007-11-01

    We present a detailed description of a theory and a program called 3P. "3P" stands for periodicity, planarity, and pixel. The 3P program is based on the intrinsic periodic correlations between residual dipolar couplings (RDCs) and in-plane internuclear vectors, and between RDCs and the orientation of peptide planes relative to an alignment tensor. The program extracts accurate rhombic, axial components of the alignment tensor without explicit coordinates, and discrete peptide plane orientations, which are utilized in combination with readily available phi/psi angles to determine the three-dimensional backbone structures of proteins. The 3P program uses one alignment tensor. We demonstrate the utility and robustness of the program, using both experimental and synthetic data sets, which were added with different levels of noise or were incomplete. The program is interfaced to Xplor-NIH via a "3P" module and is available to the public. The limitations and differences between our program and existing methods are also discussed. PMID:17892961

  5. The backbone of a city

    NASA Astrophysics Data System (ADS)

    Scellato, S.; Cardillo, A.; Latora, V.; Porta, S.

    2006-03-01

    Recent studies have revealed the importance of centrality measures to analyze various spatial factors affecting human life in cities. Here we show how it is possible to extract the backbone of a city by deriving spanning trees based on edge betweenness and edge information. By using as sample cases the cities of Bologna and San Francisco, we show how the obtained trees are radically different from those based on edge lengths, and allow an extended comprehension of the “skeleton” of most important routes that so much affects pedestrian/vehicular flows, retail commerce vitality, land-use separation, urban crime and collective dynamical behaviours.

  6. Effects of phosphorylation on the intrinsic propensity of backbone conformations of serine/threonine.

    PubMed

    He, Erbin; Yan, Guanghui; Zhang, Jian; Wang, Jun; Li, Wenfei

    2016-03-01

    Each amino acid has its intrinsic propensity for certain local backbone conformations, which can be further modulated by the physicochemical environment and post-translational modifications. In this work, we study the effects of phosphorylation on the intrinsic propensity for different local backbone conformations of serine/threonine by molecular dynamics simulations. We showed that phosphorylation has very different effects on the intrinsic propensity for certain local backbone conformations for the serine and threonine. The phosphorylation of serine increases the propensity of forming polyproline II, whereas that of threonine has the opposite effect. Detailed analysis showed that such different responses to phosphorylation mainly arise from their different perturbations to the backbone hydration and the geometrical constraints by forming side-chain-backbone hydrogen bonds due to phosphorylation. Such an effect of phosphorylation on backbone conformations can be crucial for understanding the molecular mechanism of phosphorylation-regulated protein structures/dynamics and functions. PMID:26759163

  7. Transition-metal prion protein attachment: Competition with copper

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Bernholc, Jerry

    2012-02-01

    Prion protein, PrP, is a protein capable of binding copper ions in multiple modes depending on their concentration. Misfolded PrP is implicated in a group of neurodegenerative diseases, which include ``mad cow disease'' and its human form, variant Creutzfeld-Jacob disease. An increasing amount of evidence suggests that attachment of non-copper metal ions to PrP triggers transformations to abnormal forms similar to those observed in prion diseases. In this work, we use hybrid Kohn-Sham/orbital-free density functional theory simulations to investigate copper replacement by other transition metals that bind to PrP, including zinc, iron and manganese. We consider all known copper binding modes in the N-terminal domain of PrP. Our calculations identify modes most susceptible to copper replacement and reveal metals that can successfully compete with copper for attachment to PrP.

  8. Multistability and dynamic transitions of intracellular Min protein patterns.

    PubMed

    Wu, Fabai; Halatek, Jacob; Reiter, Matthias; Kingma, Enzo; Frey, Erwin; Dekker, Cees

    2016-01-01

    Cells owe their internal organization to self-organized protein patterns, which originate and adapt to growth and external stimuli via a process that is as complex as it is little understood. Here, we study the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions. De novo formation of patterns from homogenous starting conditions is observed and studied both experimentally and in simulations. A new theoretical approach is developed for probing pattern stability under perturbations. Quantitative experiments and simulations show that, once established, Min oscillations tolerate a large degree of intracellular heterogeneity, allowing distinctly different patterns to persist in different cells with the same geometry. Min patterns maintain their axes for hours in experiments, despite imperfections, expansion, and changes in cell shape during continuous cell growth. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. The instances of multistability studied here are the combined outcome of boundary growth and strongly nonlinear kinetics, which are characteristic of the reaction-diffusion patterns that pervade biology at many scales. PMID:27279643

  9. The "universal polymer backbone" concept

    NASA Astrophysics Data System (ADS)

    Pollino, Joel Matthew

    This thesis begins with a brief analysis of the synthetic methodologies utilized in polymer science. A conclusion is drawn inferring that upper limits in molecular design are inevitable, arising as a direct consequence of the predominance of covalent strategies in the field. To address these concerns, the 'universal polymer backbone' (UPB) concept has been hypothesized. A UPB has been defined as any copolymer, side-chain functionalized with multiple recognition elements that are individually capable of forming strong, directional, and reversible non-covalent bonds. Non-covalent functionalization of these scaffolds can lead to the formation of a multitude of new polymer structures, each stemming from a single parent or 'universal polymer backbone'. To prepare such a UPB, isomerically pure exo-norbornene esters containing either a PdII SCS pincer complex or a diaminopyridine residue were synthesized, polymerized, and copolymerized via ROMP. All polymerizations were living under mild reaction conditions. Kinetic studies showed that the kp values are highly dependent upon the isomeric purity but completely independent of the terminal recognition units. Non-covalent functionalization of these copolymers was accomplished via (1) directed self-assembly, (2) multi-step self-assembly , and (3) one-step orthogonal self-assembly. This system shows complete specificity of each recognition motif for its complementary unit with no observable changes in the association constant upon functionalization. To explore potential applications of this UPB concept, random terpolymers possessing high concentrations of pendant alkyl chains and small amounts of recognition units were synthesized. Non-covalent crosslinking using a directed functionalization strategy resulted in dramatic increases in solution viscosities for metal crosslinked polymers with only minor changes in viscosity for hydrogen bonding motifs. The crosslinked materials were further functionalized via self-assembly by

  10. From Helix–Coil Transitions to Protein Folding

    PubMed Central

    Scheraga, Harold A.

    2009-01-01

    An evolution of procedures to simulate protein structure and folding pathways is described. From an initial focus on the helix–coil transition and on hydrogen-bonding and hydrophobic interactions, our original attempts to determine protein structure and folding pathways were based on an experimental approach. Experiments on the oxidative folding of reduced bovine pancreatic ribonuclease A (RNase A) led to a mechanism by which the molecule folded to the native structure by a minimum of four different pathways. The experiments with RNase A were followed by development of a molecular mechanics approach, first, making use of global optimization procedures and then with molecular dynamics (MD), evolving from an all-atom to a united-residue model. This hierarchical MD approach facilitated probing of the folding trajectory to longer time scales than with all-atom MD, and hence led to the determination of complete folding trajectories, thus far for a protein containing as many as 75 amino acid residues. With increasing refinement of the computational procedures, the computed results are coming closer to experimental observations, providing an understanding as to how physics directs the folding process. PMID:18008324

  11. Flexible backbone aromatic polyimide adhesives

    NASA Technical Reports Server (NTRS)

    Progar, Donald J.; St.clair, Terry L.

    1988-01-01

    Continuing research at Langley Research Center on the synthesis and development of new inexpensive flexible aromatic polyimides as adhesives has resulted in a material identified as LARC-F-SO2 with similarities to polyimidesulfone, PISO2, and other flexible backbone polyimides recently reported by Progar and St. Clair. Also prepared and evaluated was an endcapped version of PISO2. These two polymers were compared with LARC-TPI and LARC-STPI, polyimides research in our laboratory and reported in the literature. The adhesive evaluation, primarily based on lap shear strength (LSS) tests at RT, 177 C and 204 C, involved preparing adhesive tapes, conducting bonding studies and exposing lap shear specimens to 204 C air for up to 1000 hrs and to a 72-hour water boil. The type of adhesive failure as well as the Tg was determined for the fractured specimens. The results indicate that LARC-TPI provides the highest LSSs. LARC-F-SO2, LARC-TPI and LARC-STPI all retain their strengths after thermal exposure for 1000 hrs and PISO2 retains greater than 80 percent of its control strengths. After a 72-hr water boil exposure, most of the four adhesive systems showed reduced strengths for all test temperatures although still retaining a high percentage of their original strength (greater than 60 percent) except for one case. The predominant failure type was cohesive with no significant change in the Tgs.

  12. Flexible backbone aromatic polyimide adhesives

    NASA Technical Reports Server (NTRS)

    Progar, Donald J.; St. Clair, Terry L.

    1989-01-01

    Continuing research at Langley Research Center on the synthesis and development of new inexpensive flexible aromatic polyimides as adhesives has resulted in a material identified as LARC-F-SO2 with similarities to polyimidesulfone, PISO2, and other flexible backbone polyimides recently reported by Progar and St. Clair. Also prepared and evaluated was an endcapped version of PISO2. These two polymers were compared with LARC-TPI and LARC-STPI, polyimides research in our laboratory and reported in the literature. The adhesive evaluation, primarily based on lap shear strength (LSS) tests at RT, 177 C and 204 C, involved preparing adhesive tapes, conducting bonding studies and exposing lap shear specimens to 204 C air for up to 1000 hrs and to a 72-hour water boil. The type of adhesive failure as well as the Tg was determined for the fractured specimens. The results indicate that LARC-TPI provides the highest LSSs. LARC-F-SO2, LARC-TPI and LARC-STPI all retain their strengths after thermal exposure for 1000 hrs and PISO2 retains greater than 80 percent of its control strengths. After a 72-hr water boil exposure, most of the four adhesive systems showed reduced strengths for all test temperatures although still retaining a high percentage of their original strength (greater than 60 percent) except for one case. The predominant failure type was cohesive with no significant change in the Tgs.

  13. Free backbone carbonyls mediate rhodopsin activation.

    PubMed

    Kimata, Naoki; Pope, Andreyah; Sanchez-Reyes, Omar B; Eilers, Markus; Opefi, Chikwado A; Ziliox, Martine; Reeves, Philip J; Smith, Steven O

    2016-08-01

    Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins. PMID:27376589

  14. Glass transitions in aqueous solutions of protein (bovine serum albumin).

    PubMed

    Shinyashiki, Naoki; Yamamoto, Wataru; Yokoyama, Ayame; Yoshinari, Takeo; Yagihara, Shin; Kita, Rio; Ngai, K L; Capaccioli, Simone

    2009-10-29

    Measurements by adiabatic calorimetry of heat capacities and enthalpy relaxation rates of a 20% (w/w) aqueous solution of bovine serum albumin (BSA) by Kawai, Suzuki, and Oguni [Biophys. J. 2006, 90, 3732] have found several enthalpy relaxations at long times indicating different processes undergoing glass transitions. In a quenched sample, one enthalpy relaxation at around 110 K and another over a wide temperature range (120-190 K) were observed. In a sample annealed at 200-240 K after quenching, three separated enthalpy relaxations at 110, 135, and above 180 K were observed. Dynamics of processes probed by adiabatic calorimetric data are limited to long times on the order of 10(3) s. A fuller understanding of the processes can be gained by probing the dynamics over a wider time/frequency range. Toward this goal, we performed broadband dielectric measurements of BSA-water mixtures at various BSA concentrations over a wide frequency range of thirteen decades from 2 mHz to 1.8 GHz at temperatures from 80 to 270 K. Three relevant relaxation processes were detected. For relaxation times equal to 100 s, the three processes are centered approximately at 110, 135, and 200 K, in good agreement with those observed by adiabatic calorimetry. We have made the following interpretation of the molecular origins of the three processes. The fastest relaxation process having relaxation time of 100 or 1000 s at ca. 110 K is due to the secondary relaxation of uncrystallized water (UCW) in the hydration shell. The intermediate relaxation process with 100 s relaxation time at ca. 135 K is due to ice. The slowest relaxation process having relaxation time of 100 s at ca. 200 K is interpreted to originate from local chain conformation fluctuations of protein slaved by water. Experimental evidence supporting these interpretations include the change of temperature dependence of the relaxation time of the UCW at approximately T(gBSA) approximately = 200 K, the glass transition temperature of

  15. Transitive closure and metric inequality of weighted graphs:detecting protein interaction modules using cliques

    SciTech Connect

    Ding, Chris; He, Xiaofeng; Xiong, Hui; Peng, Hanchuan; Holbrook,Stephen R.

    2006-06-02

    We study transitivity properties of edge weights in complex networks. We show that enforcing transitivity leads to a transitivity inequality which is equivalent to ultra-metric inequality. This can be used to define transitive closure on weighted undirected graphs, which can be computed using a modified Floyd-Warshall algorithm. We outline several applications and present results of detecting protein functional modules in a protein interaction network.

  16. Pendant Dynamics of Ethylene-Oxide Containing Polymers with Diverse Backbones

    NASA Astrophysics Data System (ADS)

    Bartels, Joshua; Wang, Jing-Han Helen; Chen, Quan; Runt, James; Colby, Ralph

    In the last twenty years, a wide variety of ion conducting polymers have used ether oxygens to facilitate ion conduction, and it is therefore important to understand the dynamics of ether oxygens (EOs) when attached to different polymer backbones. Four different EO-containing polymer architectures are studied by dielectric spectroscopy to understand the backbone effect on the EO dipoles. Polysiloxanes, polyphosphazenes, polymethylmethacrylates, and a polyester ether are compared, with different EO pendant lengths for the siloxane and methylmethacrylate backbones. The flexible polysiloxanes and polyphosphazene backbones impart superior segmental mobility with a glass transition temperature 15 K lower than that of the organic backbone polymers. Short EO pendants are found to impart a lower static dielectric constant at comparable EO content as compared to longer EO pendants of either inorganic or organic backbones. The long-pendant polymethylmethacrylate polymers show two relaxations corresponding to fast EOs near the pendant tail end and slow EOs close to the slower backbone, whereas the long-pendant polysiloxane shows a single relaxation due to the siloxane backbone relaxing faster than the EO pendant. Supported by the NSF Division of Materials Research Polymers Program through Grants DMR-1404586 (RHC) and DMR-1505953 (JR).

  17. Statistical Mechanical Theory of Protein Conformation and Its Transition

    NASA Astrophysics Data System (ADS)

    Kobayashi, Yukio; Wako, Hiroshi; Saitô, Nobuhiko

    2007-07-01

    The statistical mechanical theory of the structural transitions of proteins is developed in accordance with the island model by considering the hydrophobic interactions and the entropy factors while connecting the two hydrophobic residues. The proteins treated here are apo-α-lactalbumin (1B9O), lysozyme (1LZ1), ferrocytochrome c (1CYC), cytochrome c (isozyme 1) (1YCC), chymotrypsin inhibitor 2 (2CI2), and ubiquitin (1UBQ). Among them, according to the experiments, 2CI2 and 1UBQ do not exhibit intermediate structures (two-state model), but others do exhibit intermediate structures that are sometimes termed molten globules (three-state model). The theory related to these facts is given in terms of the island model, specifically 1B9O and 1LZ1. The stability or instability of the intermediate structures is explained by the effects of entropy during folding and the amino acid sequence. The intermediate structure is composed of several stable islands, which become unstable during unfolding.

  18. Parathyroid Hormone–Related Protein Promotes Epithelial–Mesenchymal Transition

    PubMed Central

    Ardura, Juan Antonio; Rayego-Mateos, Sandra; Rámila, David; Ruiz-Ortega, Marta

    2010-01-01

    Epithelial–mesenchymal transition (EMT) is an important process that contributes to renal fibrogenesis. TGF-β1 and EGF stimulate EMT. Recent studies suggested that parathyroid hormone–related protein (PTHrP) promotes fibrogenesis in the damaged kidney, apparently dependent on its interaction with vascular endothelial growth factor (VEGF), but whether it also interacts with TGF-β and EGF to modulate EMT is unknown. Here, PTHrP(1-36) increased TGF-β1 in cultured tubuloepithelial cells and TGF-β blockade inhibited PTHrP-induced EMT-related changes, including upregulation of α-smooth muscle actin and integrin-linked kinase, nuclear translocation of Snail, and downregulation of E-cadherin and zonula occludens-1. PTHrP(1-36) also induced EGF receptor (EGFR) activation; inhibition of protein kinase C and metalloproteases abrogated this activation. Inhibition of EGFR activation abolished these EMT-related changes, the activation of ERK1/2, and upregulation of TGF-β1 and VEGF by PTHrP(1-36). Moreover, inhibition of ERK1/2 blocked EMT induced by either PTHrP(1-36), TGF-β1, EGF, or VEGF. In vivo, obstruction of mouse kidneys led to changes consistent with EMT and upregulation of TGF-β1 mRNA, p-EGFR protein, and PTHrP. Taken together, these data suggest that PTHrP, TGF-β, EGF, and VEGF might cooperate through activation of ERK1/2 to induce EMT in renal tubuloepithelial cells. PMID:19959711

  19. External Tank - The Structure Backbone

    NASA Technical Reports Server (NTRS)

    Welzyn, Kenneth; Pilet, Jeffrey C.; Diecidue-Conners, Dawn; Worden, Michelle; Guillot, Michelle

    2011-01-01

    The External Tank forms the structural backbone of the Space Shuttle in the launch configuration. Because the tank flies to orbital velocity with the Space Shuttle Orbiter, minimization of weight is mandatory, to maximize payload performance. Choice of lightweight materials both for structure and thermal conditioning was necessary. The tank is large, and unique manufacturing facilities, tooling, handling, and transportation operations were required. Weld processes and tooling evolved with the design as it matured through several block changes, to reduce weight. Non Destructive Evaluation methods were used to assure integrity of welds and thermal protection system materials. The aluminum-lithium alloy was used near the end of the program and weld processes and weld repair techniques had to be refined. Development and implementation of friction stir welding was a substantial technology development incorporated during the Program. Automated thermal protection system application processes were developed for the majority of the tank surface. Material obsolescence was an issue throughout the 40 year program. The final configuration and tank weight enabled international space station assembly in a high inclination orbit allowing international cooperation with the Russian Federal Space Agency. Numerous process controls were implemented to assure product quality, and innovative proof testing was accomplished prior to delivery. Process controls were implemented to assure cleanliness in the production environment, to control contaminants, and to preclude corrosion. Each tank was accepted via rigorous inspections, including non-destructive evaluation techniques, proof testing, and all systems testing. In the post STS-107 era, the project focused on ascent debris risk reduction. This was accomplished via stringent process controls, post flight assessment using substantially improved imagery, and selective redesigns. These efforts were supported with a number of test programs to

  20. NET amyloidogenic backbone in human activated neutrophils.

    PubMed

    Pulze, L; Bassani, B; Gini, E; D'Antona, P; Grimaldi, A; Luini, A; Marino, F; Noonan, D M; Tettamanti, G; Valvassori, R; de Eguileor, M

    2016-03-01

    Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role. PMID:26462606

  1. Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

    SciTech Connect

    Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.; Sheri, Venkat Reddy; Kassekert, Luke A.; Chen, Shujing; Agniswamy, Johnson; Wang, Yuan-Fang; Hayashi, Hironori; Aoki, Manabu; Weber, Irene T.; Mitsuya, Hiroaki

    2015-10-30

    Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolution X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.

  2. Simple Model Study of Phase Transition Properties of Isolated and Aggregated Protein

    NASA Astrophysics Data System (ADS)

    Ji, Yong-Yun; Yi, Wei-Qi; Zhang, Lin-Xi

    2011-03-01

    We investigate the phase transition properties of isolated and aggregated protein by exhaustive numerical study in the confined conformation space with maximally compact lattice model. The study within the confined conformation space shows some general folding properties. Various sequences show different folding properties: two-state folding, three-state folding and prion-like folding behavior. We find that the aggregated protein holds a more evident transition than isolated one and the transition temperature is generally lower than that in isolated case.

  3. Solution Structure and Backbone Dynamics of Streptopain

    PubMed Central

    Wang, Chih-Chieh; Houng, Hsiang-Chee; Chen, Chun-Liang; Wang, Pei-Ju; Kuo, Chih-Feng; Lin, Yee-Shin; Wu, Jiunn-Jong; Lin, Ming T.; Liu, Ching-Chuan; Huang, Wenya; Chuang, Woei-Jer

    2009-01-01

    Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease expressed by Streptococcus pyogenes. The D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed to study the effect of the allelic variants on their protease activity. In contrast to other mutants, the G239D mutant was ∼12-fold less active. The Gly-239 residue is located within the C-terminal S230-G239 region, which cannot be observed in the x-ray structure. The three-dimensional structure and backbone dynamics of the 28-kDa mature SPE B (mSPE B) were determined. Unlike the x-ray structure of the 40-kDa zymogen SPE B (proSPE B), we observed the interactions between the C-terminal loop and the active site residues in mSPE B. The structural differences between mSPE B and proSPE B were the conformation of the C-terminal loop and the orientation of the catalytic His-195 residue, suggesting that activation and inactivation of SPE B is involved in the His-195 side-chain rotation. Dynamics analysis of mSPE B and the mSPE B/inhibitor complexes showed that the catalytic and C-terminal loops were the most flexible regions with low order parameter values of 0.5 to 0.8 and exhibited the motion on the ps/ns timescale. These findings suggest that the flexible C-terminal loop of SPE B may play an important role in controlling the substrate binding, resulting in its broad substrate specificity. PMID:19237546

  4. Transitive closure and metric inequality of weighted graphs: detecting protein interaction modules using cliques.

    PubMed

    Ding, Chris; He, Xiaofeng; Xiong, Hui; Peng, Hanchuan; Holbrook, Stephen R

    2006-01-01

    We study transitivity properties of edge weights in complex networks. We show that enforcing transitivity leads to a transitivity inequality which is equivalent to ultra-metric inequality. This can be used to define transitive closure on weighted undirected graphs, which can be computed using a modified Floyd-Warshall algorithm. These new concepts are extended to dissimilarity graphs and triangle inequalities. From this, we extend the clique concept from unweighted graph to weighted graph. We outline several applications and present results of detecting protein functional modules in a protein interaction network. PMID:18399069

  5. Identification of key residues for protein conformational transition using elastic network model

    NASA Astrophysics Data System (ADS)

    Su, Ji Guo; Jin Xu, Xian; Hua Li, Chun; Chen, Wei Zu; Wang, Cun Xin

    2011-11-01

    Proteins usually undergo conformational transitions between structurally disparate states to fulfill their functions. The large-scale allosteric conformational transitions are believed to involve some key residues that mediate the conformational movements between different regions of the protein. In the present work, a thermodynamic method based on the elastic network model is proposed to predict the key residues involved in protein conformational transitions. In our method, the key functional sites are identified as the residues whose perturbations largely influence the free energy difference between the protein states before and after transition. Two proteins, nucleotide binding domain of the heat shock protein 70 and human/rat DNA polymerase β, are used as case studies to identify the critical residues responsible for their open-closed conformational transitions. The results show that the functionally important residues mainly locate at the following regions for these two proteins: (1) the bridging point at the interface between the subdomains that control the opening and closure of the binding cleft; (2) the hinge region between different subdomains, which mediates the cooperative motions between the corresponding subdomains; and (3) the substrate binding sites. The similarity in the positions of the key residues for these two proteins may indicate a common mechanism in their conformational transitions.

  6. Statistical Analysis of RNA Backbone

    PubMed Central

    Hershkovitz, Eli; Sapiro, Guillermo; Tannenbaum, Allen; Williams, Loren Dean

    2009-01-01

    Local conformation is an important determinant of RNA catalysis and binding. The analysis of RNA conformation is particularly difficult due to the large number of degrees of freedom (torsion angles) per residue. Proteins, by comparison, have many fewer degrees of freedom per residue. In this work, we use and extend classical tools from statistics and signal processing to search for clusters in RNA conformational space. Results are reported both for scalar analysis, where each torsion angle is separately studied, and for vectorial analysis, where several angles are simultaneously clustered. Adapting techniques from vector quantization and clustering to the RNA structure, we find torsion angle clusters and RNA conformational motifs. We validate the technique using well-known conformational motifs, showing that the simultaneous study of the total torsion angle space leads to results consistent with known motifs reported in the literature and also to the finding of new ones. PMID:17048391

  7. Sequence-specific transitions of the torsion angle gamma change the polar-hydrophobic profile of the DNA grooves: implication for indirect protein-DNA recognition.

    PubMed

    Zhitnikova, Mariia Yu; Boryskina, Olena P; Shestopalova, Anna V

    2014-01-01

    Variations of the shape and polarity of the DNA grooves caused by changes of the DNA conformation play an important role in the DNA readout. Despite the fact that non-canonical trans and gauche- conformations of the DNA backbone angle γ (O5'-C5'-C4'-C3') are frequently found in the DNA crystal structures, their possible role in the DNA recognition has not been studied systematically. In order to fill in this gap, we analyze the available high-resolution crystal structures of the naked and complexed DNA. The analysis shows that the non-canonical γ angle conformations are present both in the naked and bound DNA, more often in the bound vs. naked DNA, and in the nucleotides with the A-like vs. the B-like sugar pucker. The alternative angle γ torsions are more frequently observed in the purines with the A-like sugar pucker and in the pyrimidines with the B-like sugar conformation. The minor groove of the nucleotides with non-canonical γ angle conformation is more polar, while the major groove is more hydrophobic than in the nucleotides with the classical γ torsions due to variations in exposure of the polar and hydrophobic groups of the DNA backbone. The propensity of the nucleotides with different γ angle conformations to participate in the protein-nucleic acid contacts in the minor and major grooves is connected with their sugar pucker and sequence-specific. Our findings imply that the angle γ transitions contribute to the process of the protein-DNA recognition due to modification of the polar/hydrophobic profile of the DNA grooves. PMID:23998351

  8. AbDesign: an algorithm for combinatorial backbone design guided by natural conformations and sequences

    PubMed Central

    Lapidoth, Gideon D.; Baran, Dror; Pszolla, Gabriele M.; Norn, Christoffer; Alon, Assaf; Tyka, Michael D.; Fleishman, Sarel J.

    2016-01-01

    Computational design of protein function has made substantial progress, generating new enzymes, binders, inhibitors, and nanomaterials not previously seen in nature. However, the ability to design new protein backbones for function – essential to exert control over all polypeptide degrees of freedom – remains a critical challenge. Most previous attempts to design new backbones computed the mainchain from scratch. Here, instead, we describe a combinatorial backbone and sequence optimization algorithm called AbDesign, which leverages the large number of sequences and experimentally determined molecular structures of antibodies to construct new antibody models, dock them against target surfaces and optimize their sequence and backbone conformation for high stability and binding affinity. We used the algorithm to produce antibody designs that target the same molecular surfaces as nine natural, high-affinity antibodies; in six the backbone conformation at the core of the antibody binding surface is similar to the natural antibody targets, and in several cases sequence and sidechain conformations recapitulate those seen in the natural antibodies. In the case of an anti-lysozyme antibody, designed antibody CDRs at the periphery of the interface, such as L1 and H2, show a greater backbone conformation diversity than the CDRs at the core of the interface, and increase the binding surface area compared to the natural antibody, which could enhance affinity and specificity. PMID:25670500

  9. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

    PubMed Central

    Craige, Branch; Tsao, Che-Chia; Diener, Dennis R.; Hou, Yuqing; Lechtreck, Karl-Ferdinand; Rosenbaum, Joel L.

    2010-01-01

    Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition. PMID:20819941

  10. WDM backbone network with guaranteed performance planning

    NASA Astrophysics Data System (ADS)

    Liang, Peng; Sheng, Wang; Zhong, Xusi; Li, Lemin

    2005-11-01

    Wavelength-Division multiplexing (WDM), which allows a single fibre to carry multiple signals simultaneously, has been widely used to increase link capacity and is a promising technology in backbone transport network. But designing such WDM backbone network is hard for two reasons, one is the uncertainty of future traffic demand, the other is difficulty of planning of the backup resource for failure conditions. As a result, enormous amount of link capacity for the network has to be provided for the network. Recently, a new approach called Valiant Load-Balanced Scheme (VLBS) has been proposed to design the WDM backbone network. The network planned by Valiant Load-Balanced Scheme is insensitive to the traffic and continues to guarantee performance under a user defined number of link or node failures. In this paper, the Valiant Load-Balanced Scheme (VLBS) for backbone network planning has been studied and a new Valiant Load-Balanced Scheme has been proposed. Compared with the early work, the new Valiant Load-Balanced Scheme is much more general and can be used for the computation of the link capacity of both homogeneous and heterogeneous networks. The abbreviation for the general Valiant Load-Balanced Scheme is GVLBS. After a brief description of the VLBS, we will give the detail derivation of the GVLBS. The central concept of the derivation of GVLBS is transforming the heterogeneous network into a homogeneous network, and taking advantage of VLBS to get GVLBS. Such transformation process is described and the derivation and analysis of GVLBS for link capacity under normal and failure conditions is also given. The numerical results show that GVLBS can compute the minimum link capacity required for the heterogeneous backbone network under different conditions (normal or failure).

  11. Conformational transition in signal transduction: metastable states and transition pathways in the activation of a signaling protein.

    PubMed

    Banerjee, Rahul; Yan, Honggao; Cukier, Robert I

    2015-06-01

    Signal transduction is of vital importance to the growth and adaptation of living organisms. The key to understand mechanisms of biological signal transduction is elucidation of the conformational dynamics of its signaling proteins, as the activation of a signaling protein is fundamentally a process of conformational transition from an inactive to an active state. A predominant form of signal transduction for bacterial sensing of environmental changes in the wild or inside their hosts is a variety of two-component systems, in which the conformational transition of a response regulator (RR) from an inactive to an active state initiates responses to the environmental changes. Here, RR activation has been investigated using RR468 as a model system by extensive unbiased all-atom molecular dynamics (MD) simulations in explicit solvent, starting from snapshots along a targeted MD trajectory that covers the conformational transition. Markov state modeling, transition path theory, and geometric analyses of the wealth of the MD data have provided a comprehensive description of the RR activation. It involves a network of metastable states, with one metastable state essentially the same as the inactive state and another very similar to the active state that are connected via a small set of intermediates. Five major pathways account for >75% of the fluxes of the conformational transition from the inactive to the active-like state. The thermodynamic stability of the states and the activation barriers between states are found, to identify rate-limiting steps. The conformal transition is initiated predominantly by movements of the β3α3 loop, followed by movements of the β4α4-loop and neighboring α4 helix region, and capped by additional movements of the β3α3 loop. A number of transient hydrophobic and hydrogen bond interactions are revealed, and they may be important for the conformational transition. PMID:25945797

  12. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  13. Unraveling protein stabilization mechanisms: vitrification and water replacement in a glass transition temperature controlled system.

    PubMed

    Grasmeijer, N; Stankovic, M; de Waard, H; Frijlink, H W; Hinrichs, W L J

    2013-04-01

    The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominantly determines stability at higher glass transition temperatures. PMID:23360765

  14. Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly

    PubMed Central

    Horne, W. Seth; Price, Joshua L.; Gellman, Samuel H.

    2008-01-01

    The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the α prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins. PMID:18587049

  15. Protein palmitoylation activate zygotic gene expression during the maternal-to-zygotic transition.

    PubMed

    Du, Zhaoxia; Chen, Xueran; Li, Xian; He, Kun; Ji, Shufang; Shi, Wei; Hao, Aijun

    2016-06-24

    Upon fertilization, maternal factors direct development and trigger zygotic genome activation at the maternal-to-zygotic transition (MZT). However, the factors that activate the zygotic program in vertebrates are not well defined. Here, we found that protein palmitoylation played an important role in acquiring transcriptional competency and orchestrating the clearance of the maternal program in zebrafish. After inhibition of protein palmitoylation, zebrafish embryos developed normally before the Mid-Blastula Transition (MBT); however, they did not initiate epiboly. Moreover, our results showed that protein palmitoylation is required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression. PMID:27235108

  16. Conformational Transitions in Protein-Protein Association: Binding of Fasciculin-2 to Acetylcholinesterase

    PubMed Central

    Bui, Jennifer M.; Radic, Zoran; Taylor, Palmer; McCammon, J. Andrew

    2006-01-01

    The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex. PMID:16473897

  17. Membrane tension and peripheral protein density mediate membrane shape transitions

    PubMed Central

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation, and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature coupling proteins and therefore a wide range of endocytic proteins. PMID:25569184

  18. Membrane tension and peripheral protein density mediate membrane shape transitions.

    PubMed

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins. PMID:25569184

  19. Membrane tension and peripheral protein density mediate membrane shape transitions

    NASA Astrophysics Data System (ADS)

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins.

  20. ANSS Backbone Station Installation and Site Characterization

    NASA Astrophysics Data System (ADS)

    Meremonte, M.; Leeds, A.; Overturf, D.; McMillian, J.; Allen, J.; McNamara, D.

    2004-12-01

    During 2004 several new broadband seismic stations have been deployed as a part of the USGS's Advanced National Seismic System (ANSS) backbone and regional networks. New stations include: ERPA, MNTX, OGLA, AMTX, NATX, KCCO, BMO, MARC, TZTN, LAO, DGMT, REDW, KSU1, MOOW, TPAW, LOHW, RAMW. Permanent station locations were chosen to minimize the local noise conditions by recording continuous data and using a quantitative analysis of the statistical distribution of noise power estimates. For each one-hour segment of continuous data, a power spectral density (PSD) is estimated and smoothed in full octave averages at 1/8 octave intervals. Powers for each 1/8 period interval were then accumulated in one dB power bins. A statistical analysis of power bins yields probability density functions (PDFs) as a function of noise power for each of the octave bands at each station and component. Examination of earthquake signal, artifacts related to station operation and episodic cultural noise in the PDFs allow us to estimate both the overall station quality and the level of earth noise at each potential backbone site. The main function of a seismic network, such as the ANSS, is to provide high quality data for earthquake monitoring, source studies, and Earth structure research. The utility of seismic data is greatly increased when noise levels are reduced. A good quantification and understanding of seismic noise is a first step at reducing noise levels in seismic data and improving overall data quality from the ANSS backbone network.

  1. Max Delbruck Prize in Biological Physics Lecture: Single-molecule protein folding and transition paths

    NASA Astrophysics Data System (ADS)

    Eaton, William

    2012-02-01

    The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs by crossing the free energy barrier between two states. It is a uniquely single-molecule property, and has not yet been observed experimentally for any system in the condensed phase. The importance of the transition path in protein folding is that it contains all of the mechanistic information on how a protein folds. As a major step toward observing transition paths, we have determined the average transition-path time for a fast and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule FRET experiments. While the folding rate coefficients differ by 10,000-fold, surprisingly, the transition-path times differ by less than 5-fold, showing that a successful barrier crossing event takes almost the same time for a fast- and a slow-folding protein, i.e. almost the same time to fold when it actually happens.

  2. Ratcheted molecular-dynamics simulations identify efficiently the transition state of protein folding

    NASA Astrophysics Data System (ADS)

    Tiana, Guido; Camilloni, Carlo

    2012-12-01

    The atomistic characterization of the transition state (TS) is a fundamental step to improve the understanding of the folding mechanism and the function of proteins. From a computational point of view, the identification of the conformations that build out the transition state is particularly cumbersome, mainly because of the large computational cost of generating a statistically sound set of folding trajectories. Here we show that a biasing algorithm, based on the physics of the ratchet-and-pawl, can be used to approximate efficiently the transition state. The basic idea is that the algorithmic ratchet exerts a force on the protein when it is climbing the free-energy barrier, while it is inactive when it is descending. The transition state can be identified as the point of the trajectory where the ratchet changes regime. Besides discussing this strategy in general terms, we test it within a protein model whose transition state can be studied independently by plain molecular dynamics simulations. Finally, we show its power in explicit-solvent simulations, obtaining and characterizing a set of transition-state conformations for Acyl-Coenzyme A-Binding Protein (ACBP) and Chymotrypsin Inhibitor 2 (CI2).

  3. Phase Transitions in the Assembly of Multi-Valent Signaling Proteins

    PubMed Central

    Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun; Kim, Soyeon; Chen, Baoyu; Guo, Liang; Llaguno, Marc; Hollingsworth, Javoris V.; King, David S.; Banani, Salman F.; Russo, Paul S.; Jiang, Qiu-Xing; Nixon, B. Tracy; Rosen, Michael K.

    2012-01-01

    Cells are organized on length scales ranging from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are not well understood. Here we show that interactions between diverse, synthetic multivalent macromolecules (including multi-domain proteins and RNA) produce sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its established biological partners Nck and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology. PMID:22398450

  4. Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

    PubMed Central

    Taylor, Randall S.; Jones, Steven M.; Dahl, Rolf H.; Nordeen, Mark H.; Howell, Kathryn E.

    1997-01-01

    To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen. PMID:9348533

  5. Direct observation of transition paths during the folding of proteins and nucleic acids.

    PubMed

    Neupane, Krishna; Foster, Daniel A N; Dee, Derek R; Yu, Hao; Wang, Feng; Woodside, Michael T

    2016-04-01

    Transition paths, the fleeting trajectories through the transition states that dominate the dynamics of biomolecular folding reactions, encapsulate the critical information about how structure forms. Owing to their brief duration, however, they have not previously been observed directly. We measured transition paths for both nucleic acid and protein folding, using optical tweezers to observe the microscopic diffusive motion of single molecules traversing energy barriers. The average transit times and the shapes of the transit-time distributions agreed well with theoretical expectations for motion over the one-dimensional energy landscapes reconstructed for the same molecules, validating the physical theory of folding reactions. These measurements provide a first look at the critical microscopic events that occur during folding, opening exciting new avenues for investigating folding phenomena. PMID:27124461

  6. Analysis of Euglena gracilis Plastid-Targeted Proteins Reveals Different Classes of Transit Sequences▿

    PubMed Central

    Durnford, Dion G.; Gray, Michael W.

    2006-01-01

    The plastid of Euglena gracilis was acquired secondarily through an endosymbiotic event with a eukaryotic green alga, and as a result, it is surrounded by a third membrane. This membrane complexity raises the question of how the plastid proteins are targeted to and imported into the organelle. To further explore plastid protein targeting in Euglena, we screened a total of 9,461 expressed sequence tag (EST) clusters (derived from 19,013 individual ESTs) for full-length proteins that are plastid localized to characterize their targeting sequences and to infer potential modes of translocation. Of the 117 proteins identified as being potentially plastid localized whose N-terminal targeting sequences could be inferred, 83 were unique and could be classified into two major groups. Class I proteins have tripartite targeting sequences, comprising (in order) an N-terminal signal sequence, a plastid transit peptide domain, and a predicted stop-transfer sequence. Within this class of proteins are the lumen-targeted proteins (class IB), which have an additional hydrophobic domain similar to a signal sequence and required for further targeting across the thylakoid membrane. Class II proteins lack the putative stop-transfer sequence and possess only a signal sequence at the N terminus, followed by what, in amino acid composition, resembles a plastid transit peptide. Unexpectedly, a few unrelated plastid-targeted proteins exhibit highly similar transit sequences, implying either a recent swapping of these domains or a conserved function. This work represents the most comprehensive description to date of transit peptides in Euglena and hints at the complex routes of plastid targeting that must exist in this organism. PMID:16998072

  7. High throughput methods for analyzing transition metals in proteins on a microgram scale.

    PubMed

    Atanassova, Anelia; Högbom, Martin; Zamble, Deborah B

    2008-01-01

    Transition metals are among the most common ligands that contribute to the biochemical and physiological properties of proteins. In the course of structural proteomic projects, the detection of transition metal cofactors prior to the determination of a high-resolution structure is extremely beneficial. This information can be used to select tractable targets from the proteomic pipeline because the presence of a metal often improves protein stability and can be used to help solve the phasing problem in x-ray crystallography. Recombinant proteins are often purified with substoichiometric amounts of metal loaded, so additional metal may be needed to obtain the homogeneous protein solution crucial for structural analysis. Furthermore, identifying a metal cofactor provides a clue about the nature of the biological role of an unclassified protein and can be applied with structural data in the assignation of a putative function. Many of the existing methods for transition metal analysis of purified proteins have limitations, which include a requirement for a large quantity of protein or a reliance on equipment with a prohibitive cost.The authors have developed two simple high throughput methods for identifying metalloproteins on a microgram scale. Each of the techniques has distinct advantages and can be applied to address divergent experimental goals. The first method, based on simple luminescence and colorimetric reactions, is fast, cheap, and semiquantitative. The second method, which employs HPLC separation, is accurate and affords unambiguous metal identification. PMID:18542873

  8. Transitions.

    ERIC Educational Resources Information Center

    Nathanson, Jeanne H., Ed.

    1993-01-01

    This theme issue on transitions for individuals with disabilities contains nine papers discussing transition programs and issues. "Transition Issues for the 1990s," by Michael J. Ward and William D. Halloran, discusses self-determination, school responsibility for transition, continued educational engagement of at-risk students, and service…

  9. Conservation of Transit Peptide-Independent Protein Import into the Mitochondrial and Hydrogenosomal Matrix

    PubMed Central

    Garg, Sriram; Stölting, Jan; Zimorski, Verena; Rada, Petr; Tachezy, Jan; Martin, William F.; Gould, Sven B.

    2015-01-01

    The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import. PMID:26338186

  10. Base-pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA-coat protein complex.

    PubMed

    Boyce, Michael; Scott, Felicia; Guogas, Laura M; Gehrke, Lee

    2006-01-01

    The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function. PMID:16312015

  11. Kinetic Definition of Protein Folding Transition State Ensembles and Reaction Coordinates

    PubMed Central

    Snow, Christopher D.; Rhee, Young Min; Pande, Vijay S.

    2006-01-01

    Using distributed molecular dynamics simulations we located four distinct folding transitions for a 39-residue ββαβ protein fold. To characterize the nature of each room temperature transition, we calculated the probability of transmission for 500 points along each free energy barrier. We introduced a method for determining transition states by employing the transmission probability, Ptrans, and determined which conformations were transition state ensemble members (Ptrans ≈ 0.5). The transmission probability may be used to characterize the barrier in several ways. For example, we ran simulations at 82°C, determined the change in Ptrans with temperature for all 2,000 conformations, and quantified Hammond behavior directly using Ptrans correlation. Additionally, we propose that diffusion along Ptrans may provide the configurational diffusion rate at the top of the barrier. Specifically, given a transition state conformation x0 with estimated Ptrans = 0.5, we selected a large set of subsequent conformations from independent trajectories, each exactly a small time δt after x0 (250 ps). Calculating Ptrans for the new trial conformations, we generated the P(Ptrans|δt = 250 ps) distribution that reflected diffusion. This approach provides a novel perspective on the diffusive nature of a protein folding transition and provides a framework for a quantitative study of activated relaxation kinetics. PMID:16617068

  12. Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins

    PubMed Central

    Whitten, Steven T.; García-Moreno E., Bertrand; Hilser, Vincent J.

    2005-01-01

    Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are important determinants of the cooperativity of ligand-driven global structural transitions, and (iii) are well represented thermodynamically as local unfolding processes. These studies illustrate how an ensemble-based description of proteins can be used to describe quantitatively the interdependence of local conformational fluctuations, ligand-binding processes, and global structural transitions. This level of understanding of the relationship between conformation, energy, and dynamics is required for a detailed mechanistic understanding of allostery, cooperativity, and other complex functional and regulatory properties of macromolecules. PMID:15767576

  13. Coarse-Grained Simulations of Heme Proteins: Validation and Study of Large Conformational Transitions.

    PubMed

    Ramírez, Claudia L; Petruk, Ariel; Bringas, Mauro; Estrin, Dario A; Roitberg, Adrian E; Marti, Marcelo A; Capece, Luciana

    2016-07-12

    Heme proteins are ubiquitous in nature and perform many diverse functions in all kingdoms of life. Many of these functions are related to large-scale conformational transitions and allosteric processes. Sampling of these large conformational changes is computationally very challenging. In this context, coarse-grain simulations emerge as an efficient approach to explore the conformational landscape. In this work, we present a coarse-grained model of the heme group and thoroughly validate this model in different benchmark examples, which include the monomeric heme proteins myoglobin and neuroglobin and the tetrameric human hemoglobin where we evaluated the method's ability to explore conformational changes (as the formation of hexacoordinated species) and allosteric transitions (as the well-known R → T transition). The obtained results are compared with atomistic molecular dynamics simulations. Overall, the results indicate that this approach conserves the essential dynamical information on different allosteric processes. PMID:27267322

  14. Transitions.

    ERIC Educational Resources Information Center

    Field, David; And Others

    1992-01-01

    Includes four articles: "Career Aspirations" (Field); "Making the Transition to a New Curriculum" (Baker, Householder); "How about a 'Work to School' Transition?" (Glasberg); and "Technological Improvisation: Bringing CNC to Woodworking" (Charles, McDuffie). (SK)

  15. Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

    PubMed

    Vermouth, N T; Carriazo, C S; Ponce, R H; Blanco, A

    1986-01-01

    Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda. PMID:3956158

  16. Transition.

    ERIC Educational Resources Information Center

    Thompson, Sandy, Ed.; And Others

    1990-01-01

    This "feature issue" focuses on transition from school to adult life for persons with disabilities. Included are "success stories," brief program descriptions, and a list of resources. Individual articles include the following titles and authors: "Transition: An Energizing Concept" (Paul Bates); "Transition Issues for the 1990s" (William Halloran…

  17. Core-Shell Model of Folding-Unfolding Transitions (UFT) in Proteins

    NASA Astrophysics Data System (ADS)

    Aroutiounian, Svetlana

    2008-03-01

    There are ˜10^N conformations for a protein of length N to sort out randomly in search of lowest free energy state. Can protein folding be simple and fast? Core-shell model introduces principles, proposes mechanisms and scores residues of fast, reversible UFT in protein. According to it, during UFT the realm of intra-residual interactions leads the residue motion. The scaffold of hydrophilic residues forms external shell of unstructured, tube-like protein in unfolded state, just as the hydrophobic residues form internal scaffold -- core, of the protein in folded state. As UFT proceeds, residue slides into lowest-score position permitted by its structure. Model accounts for experimentally observed features of UFT. It is based on three principles: 1) During UFT protein is virtual - its features or structure are inferred only statistically and with limited precision; 2) Mechanism of UFT memory is not longitudinal, but transverse; 3) Native design overrides specific features of residues - the alphabet of amino acids assumes an intrinsic score-function. Per-residue mechanism of UFT is proposed and score-function is described. Difference graphs of transitional score-function and average genome-wide abundance index show that our score-function is the order parameter of UFT in protein and by virtue of being it, reveals transitional key residues. It echoes the multiple-tier and funnel concepts of FEL perspective. Monte Carlo simulations of UFT in myoglobin illustrate the idea.

  18. A high-performance liquid chromatography method for determining transition metal content in proteins.

    PubMed

    Atanassova, Anelia; Lam, Robert; Zamble, Deborah B

    2004-12-01

    Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed. PMID:15519577

  19. Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.

    PubMed Central

    Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime

    2014-01-01

    Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted ‘silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers. PMID:25004800

  20. Differential Scanning Fluorimetry provides high throughput data on silk protein transitions.

    NASA Astrophysics Data System (ADS)

    Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime

    2014-07-01

    Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted `silk' fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers.

  1. Protein accumulation in the endoplasmic reticulum as a non-equilibrium phase transition.

    PubMed

    Budrikis, Zoe; Costantini, Giulio; La Porta, Caterina A M; Zapperi, Stefano

    2014-01-01

    Several neurological disorders are associated with the aggregation of aberrant proteins, often localized in intracellular organelles such as the endoplasmic reticulum. Here we study protein aggregation kinetics by mean-field reactions and three dimensional Monte carlo simulations of diffusion-limited aggregation of linear polymers in a confined space, representing the endoplasmic reticulum. By tuning the rates of protein production and degradation, we show that the system undergoes a non-equilibrium phase transition from a physiological phase with little or no polymer accumulation to a pathological phase characterized by persistent polymerization. A combination of external factors accumulating during the lifetime of a patient can thus slightly modify the phase transition control parameters, tipping the balance from a long symptomless lag phase to an accelerated pathological development. The model can be successfully used to interpret experimental data on amyloid-β clearance from the central nervous system. PMID:24722051

  2. Protein accumulation in the endoplasmic reticulum as a non-equilibrium phase transition

    PubMed Central

    Budrikis, Zoe; Costantini, Giulio; La Porta, Caterina A. M.; Zapperi, Stefano

    2014-01-01

    Several neurological disorders are associated with the aggregation of aberrant proteins, often localized in intracellular organelles such as the endoplasmic reticulum. Here we study protein aggregation kinetics by mean-field reactions and three dimensional Monte carlo simulations of diffusion-limited aggregation of linear polymers in a confined space, representing the endoplasmic reticulum. By tuning the rates of protein production and degradation, we show that the system undergoes a non-equilibrium phase transition from a physiological phase with little or no polymer accumulation to a pathological phase characterized by persistent polymerization. A combination of external factors accumulating during the lifetime of a patient can thus slightly modify the phase transition control parameters, tipping the balance from a long symptomless lag phase to an accelerated pathological development. The model can be successfully used to interpret experimental data on amyloid-β clearance from the central nervous system. PMID:24722051

  3. Thermogelling Biodegradable Polymers with Hydrophilic Backbones: PEG-g-PLGA

    SciTech Connect

    Jeong, Byeongmoon; Kibbey, Merinda R.; Birnbaum, Jerome C.; Won, You-Yeong; Gutowska, Anna

    2000-10-31

    The aqueous solutions of poly(ethylene glycol)grafted with poly(lactic acid-co-glycolic acid) flow freely at room temperature but form gels at higher temperature. The existence of micelles in water at low polymer concentration was confirmed by Cro-transmission electron microscopy and dye solubilization studies. The micellar diameter and critical micelle concentration are about 9 nm and 0.47 wt.% respectively. The critical gel concentration, above which a gel phase appears was 16 wt.% and sol-to-gel transition temperature was slightly affected by the concentration in the range of 16 {approx} 25 wt.%. At sol-to-gel transition, viscosity increased abruptly and C-NMR showed molecular motion of hydrophilic poly(lactic acid-co-glycolic acid) side-chains increased. The hydrogel of PEG-g-PLGA with hydrophilic backbones was transparent during degradation and remained a gel for one week, suggesting a promising material for short-term drug delivery.

  4. Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions

    PubMed Central

    Elrouby, Nabil; Bonequi, Mitzi Villajuana; Porri, Aimone; Coupland, George

    2013-01-01

    Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) plays essential roles in eukaryotic growth and development. Many covalently modified SUMO targets have been identified; however, the extent and significance of noncovalent interactions of SUMO with cellular proteins is poorly understood. Here, large-scale yeast two-hybrid screens repeatedly identified a surprisingly small number of proteins that interacted with three Arabidopsis SUMO isoforms. These SUMO-interacting proteins are nuclear and fall into two main categories: six histone or DNA methyltransferses or demethylases and six proteins that we show to be the evolutionary and functional homologs of SUMO-targeted ubiquitin ligases (STUbLs). The selectivity of the screen for several methylases and demethylases suggests that SUMO interaction with these proteins has a significant impact on chromatin methylation. Furthermore, the Arabidopsis STUbLs (AT-STUbLs) complemented to varying degrees the growth defects of the Schizosaccharomyces pombe STUbL mutant rfp1/rfp2, and three of them also complemented the genome integrity defects of this mutant, demonstrating that these proteins show STUbL activity. We show that one of the AT-STUbLs least related to the S. pombe protein, AT-STUbL4, has acquired a plant-specific function in the floral transition. It reduces protein levels of CYCLING DOF FACTOR 2, hence increasing transcript levels of CONSTANS and promoting flowering through the photoperiodic pathway. PMID:24255109

  5. Probing the Folding-Unfolding Transition of a Thermophilic Protein, MTH1880

    PubMed Central

    Jung, Youngjin; Han, Jeongmin; Yun, Ji-Hye; Chang, Iksoo; Lee, Weontae

    2016-01-01

    The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD) simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central β-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880. PMID:26766214

  6. Myelin Membrane Assembly Is Driven by a Phase Transition of Myelin Basic Proteins Into a Cohesive Protein Meshwork

    PubMed Central

    Aggarwal, Shweta; Snaidero, Nicolas; Pähler, Gesa; Frey, Steffen; Sánchez, Paula; Zweckstetter, Markus; Janshoff, Andreas; Schneider, Anja; Weil, Marie-Theres; Schaap, Iwan A. T.; Görlich, Dirk; Simons, Mikael

    2013-01-01

    Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system. PMID:23762018

  7. Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork.

    PubMed

    Aggarwal, Shweta; Snaidero, Nicolas; Pähler, Gesa; Frey, Steffen; Sánchez, Paula; Zweckstetter, Markus; Janshoff, Andreas; Schneider, Anja; Weil, Marie-Theres; Schaap, Iwan A T; Görlich, Dirk; Simons, Mikael

    2013-01-01

    Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system. PMID:23762018

  8. Responsive Gel-Gel Phase Transitions in Artificially Engineered Protein Hydrogels

    NASA Astrophysics Data System (ADS)

    Olsen, B. D.

    2012-02-01

    Artificially engineered protein hydrogels provide an attractive platform for biomedical materials due to their similarity to components of the native extracellular matrix. Engineering responsive transitions between shear-thinning and tough gel phases in these materials could potentially enable gels that are both shear-thinning and tough to be produced as novel injectable biomaterials. To engineer a gel with such transitions, a triblock copolymer with thermoresponsive polymer endblocks and an artificially engineered protein gel midblock is designed. Temperature is used to trigger a transition from a single network protein hydrogel phase to a double network phase with both protein and block copolymer networks present at different length scales. The thermodynamics of network formation and resulting structural changes are established using small-angle scattering, birefringence, and dynamic scanning calorimetry. The formation of the second network is shown to produce a large, nonlinear increase in the elastic modulus as well as enhancements in creep compliance and toughness. Although the gels show yielding behavior in both the single and double network regimes, a qualitative change in the deformation mechanism is observed due to the structural changes.

  9. Pre-transition effects mediate forces of assembly between transmembrane proteins.

    PubMed

    Katira, Shachi; Mandadapu, Kranthi K; Vaikuntanathan, Suriyanarayanan; Smit, Berend; Chandler, David

    2016-01-01

    We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order-disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to this phenomenon as the 'orderphobic effect'. The effect is mediated by proximity to the order-disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered. PMID:26910009

  10. Pre-transition effects mediate forces of assembly between transmembrane proteins

    DOE PAGESBeta

    Katira, Shachi; Mandadapu, Kranthi K.; Vaikuntanathan, Suriyanarayanan; Smit, Berend; Chandler, David

    2016-02-24

    We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to thismore » phenomenon as the 'orderphobic effect'. The effect is mediated by proximity to the order–disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered.« less

  11. Pre-transition effects mediate forces of assembly between transmembrane proteins

    PubMed Central

    Katira, Shachi; Mandadapu, Kranthi K; Vaikuntanathan, Suriyanarayanan; Smit, Berend; Chandler, David

    2016-01-01

    We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to this phenomenon as the 'orderphobic effect'. The effect is mediated by proximity to the order–disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered. DOI: http://dx.doi.org/10.7554/eLife.13150.001 PMID:26910009

  12. Backbone Assignment of the MALT1 Paracaspase by Solution NMR

    PubMed Central

    Unnerståle, Sofia; Nowakowski, Michal; Baraznenok, Vera; Stenberg, Gun; Lindberg, Jimmy; Mayzel, Maxim; Orekhov, Vladislav; Agback, Tatiana

    2016-01-01

    Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the 15N/13C/1H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins. PMID:26788853

  13. Backbone dependency further improves side chain prediction efficiency in the Energy-based Conformer Library (bEBL).

    PubMed

    Subramaniam, Sabareesh; Senes, Alessandro

    2014-11-01

    Side chain optimization is an integral component of many protein modeling applications. In these applications, the conformational freedom of the side chains is often explored using libraries of discrete, frequently occurring conformations. Because side chain optimization can pose a computationally intensive combinatorial problem, the nature of these conformer libraries is important for ensuring efficiency and accuracy in side chain prediction. We have previously developed an innovative method to create a conformer library with enhanced performance. The Energy-based Library (EBL) was obtained by analyzing the energetic interactions between conformers and a large number of natural protein environments from crystal structures. This process guided the selection of conformers with the highest propensity to fit into spaces that should accommodate a side chain. Because the method requires a large crystallographic data-set, the EBL was created in a backbone-independent fashion. However, it is well established that side chain conformation is strongly dependent on the local backbone geometry, and that backbone-dependent libraries are more efficient in side chain optimization. Here we present the backbone-dependent EBL (bEBL), whose conformers are independently sorted for each populated region of Ramachandran space. The resulting library closely mirrors the local backbone-dependent distribution of side chain conformation. Compared to the EBL, we demonstrate that the bEBL uses fewer conformers to produce similar side chain prediction outcomes, thus further improving performance with respect to the already efficient backbone-independent version of the library. PMID:25212195

  14. Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family).

    PubMed

    Kulinska, Anna; Godziszewska, Jolanta; Wojciechowska, Anna; Ludwiczak, Marta; Jagura-Burdzy, Grazyna

    2016-04-01

    The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in transnegatively interferes with mini-RA3 replication and stable maintenance of RA3. PMID:26850301

  15. Use of glass transitions in carbohydrate excipient design for lyophilized protein formulations.

    PubMed

    Roughton, Brock C; Topp, E M; Camarda, Kyle V

    2012-01-10

    This work describes an effort to apply methods from process systems engineering to a pharmaceutical product design problem, with a novel application of statistical approaches to comparing solutions. A computational molecular design framework was employed to design carbohydrate molecules with high glass transition temperatures and low water content in the maximally freeze-concentrated matrix, with the objective of stabilizing lyophilized protein formulations. Quantitative structure-property relationships were developed for glass transition temperature of the anhydrous solute, glass transition temperature of the maximally concentrated solute, melting point of ice and Gordon-Taylor constant for carbohydrates. An optimization problem was formulated to design an excipient with optimal property values. Use of a stochastic optimization algorithm, Tabu search, provided several carbohydrate excipient candidates with statistically similar property values, as indicated by prediction intervals calculated for each property. PMID:24385675

  16. Effect of surface attractive strength on structural transitions of a confined HP lattice protein

    NASA Astrophysics Data System (ADS)

    Pattanasiri, Busara; Li, Ying Wai; Wust, Thomas; Landau, David P.

    2015-09-01

    We investigate the influence of surface attractive strength on structural transitions of a hydrophobic-polar (HP) lattice protein confined in a slit formed by two parallel, attractive walls. We apply Wang-Landau sampling together with efficient Monte Carlo updates to estimate the density of states of the system. The conformational transitions, namely, the debridging process and hydrophobic core formation, can be identified by analyzing the specific heat together with several structural observables, such as the numbers of surface contacts, the number of hydrophobic pairs, and radii of gyration in different directions. As temperature decreases, we find that the occurrence of the debridging process is conditional depending on the surface attractive strength. This, in turn, affects the nature of the hydrophobic core formation that takes place at a lower temperature. We illustrate these observations with the aid of a HP protein chain with 48 monomers.

  17. Effect of surface attractive strength on structural transitions of a confined HP lattice protein

    SciTech Connect

    Pattanasiri, Busara; Li, Ying Wai; Wuest, Thomas; Landau, David P

    2015-01-01

    We investigate the influence of surface attractive strength on structural transitions of a hydrophobic-polar (HP) lattice protein confined in a slit formed by two parallel, attractive walls. We apply Wang-Landau sampling together with efficient Monte Carlo updates to estimate the density of states of the system. The conformational transitions, namely, the debridging process and hydrophobic core formation, can be identified by analyzing the specific heat together with several structural observables, such as the numbers of surface contacts, the number of hydrophobic pairs, and radii of gyration in different directions. As temperature decreases, we find that the occurrence of the debridging process is conditional depending on the surface attractive strength. This, in turn, affects the nature of the hydrophobic core formation that takes place at a lower temperature. We illustrate these observations with the aid of a HP protein chain with 48 monomers.

  18. Desmoplastic melanoma: expression of epithelial-mesenchymal transition-related proteins.

    PubMed

    Garrido, Maria Concepción; Requena, Luis; Kutzner, Heinz; Ortiz, Pablo; Pérez-Gómez, Beatriz; Rodriguez-Peralto, José-Luis

    2014-03-01

    Desmoplastic melanoma (DM) is a rare variant of melanoma. Most frequently, it seems as clinically ambiguous and histologically characterized by a poorly demarcated neoplasm composed of a proliferation of spindle melanocytes dispersed in a prominent collagenous stroma. It often represents a diagnostic challenge, delaying its detection. We analyzed the expression profile of 29 (28 "pure" and 1 "combined") DM. These data were compared with a series of 62 primary vertical growth phase nondesmoplastic melanomas (NDMs) using a set of proteins including melanocytic markers (S-100 protein and melan-A) and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, SPARC, WT1, and PKCα). The S-100 protein confirmed the melanocytic origin of the DM (positive in 96%). The significant positive expression of N-cadherin, SPARC, and WT1 in DM (61%, 82%, and 71%) compared with NDM (28%, 43%, and 47%; P < 0.05) and a lower expression of E-cadherin in DM (14%) compared with NDM (61%) support specific adhesive and migratory properties of DM tumor cells. The study was carried out with tissue microarrays that partly limited the study of the tumor sections. This study demonstrates, for the first time, a prominent expression of epithelial-mesenchymal transition-related proteins in DMs and tries to be one more step in refining its knowledge and leading to a better understanding of its biological and clinical behaviors. PMID:23974224

  19. Hydration of the folding transition-state ensemble of a protein

    PubMed Central

    Brun, Ludovic; Isom, Daniel G.; Velu, Priya; García-Moreno, Bertrand

    2008-01-01

    A complete description of the mechanisms of protein folding requires knowledge of the structural and physical character of the folding transition state ensembles (TSE). A key question remains, concerning the role of hydration of the hydrophobic core in determining folding mechanisms. To address this we probed the state of hydration of the TSE of staphylococcal nuclease (SNase) by examining the fluorescence-detected pressure-jump relaxation behavior of six SNase variants in which a residue in the hydrophobic core, Val-66, was replaced with polar or ionizable residues (Lys, Arg, His, Asp, Glu, Asn). Owing to a large positive activation volume for folding, the major effect of pressure on the wild type protein is to decrease the folding rate. By the time wild type SNase reaches the folding transition state, most water has already been expelled from its hydrophobic core. In contrast, the major effect of pressure on the variant proteins is an increase of the unfolding rate due to a large negative activation volume for unfolding. This results from a significant increase in the hydration of the TSE when an internal ionizable group is present. These data confirm that the role of water in the folding reaction can differ from protein to protein, and that even a single substitution in a critical position can modulate significantly the properties of the TSE. PMID:16533028

  20. Modulating energy arriving at photochemical reaction centers: orange carotenoid protein-related photoprotection and state transitions.

    PubMed

    Kirilovsky, Diana

    2015-10-01

    Photosynthetic organisms tightly regulate the energy arriving to the reaction centers in order to avoid photodamage or imbalance between the photosystems. To this purpose, cyanobacteria have developed mechanisms involving relatively rapid (seconds to minutes) changes in the photosynthetic apparatus. In this review, two of these processes will be described: orange carotenoid protein(OCP)-related photoprotection and state transitions which optimize energy distribution between the two photosystems. The photoactive OCP is a light intensity sensor and an energy dissipater. Photoactivation depends on light intensity and only the red-active OCP form, by interacting with phycobilisome cores, increases thermal energy dissipation at the level of the antenna. A second protein, the "fluorescence recovery protein", is needed to recover full antenna capacity under low light conditions. This protein accelerates OCP conversion to the inactive orange form and plays a role in dislodging the red OCP protein from the phycobilisome. The mechanism of state transitions is still controversial. Changes in the redox state of the plastoquinone pool induce movement of phycobilisomes and/or photosystems leading to redistribution of energy absorbed by phycobilisomes between PSII and PSI and/or to changes in excitation energy spillover between photosystems. The different steps going from the induction of redox changes to movement of phycobilisomes or photosystems remain to be elucidated. PMID:25139327

  1. Thermal Transitions and Extrusion of Glycerol-Plasticized Whey Protein Mixtures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of glycerol and moisture contents on the thermal transitions of whey protein isolate (WPI) powder-glycerol-water mixtures were studied. Mixtures with ratios of 100:0, 70:30, 60:40 and 50:50 WPI:glycerol on a dry basis (db) were pre-conditioned to 0.34+/-0.01 (25.4±0.4ºC) and 0.48+/-0.02...

  2. Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions.

    PubMed

    Glover, Karen; Mei, Yang; Sinha, Sangita C

    2016-10-01

    Many proteins contain intrinsically disordered regions (IDRs) lacking stable secondary and ordered tertiary structure. IDRs are often implicated in macromolecular interactions, and may undergo structural transitions upon binding to interaction partners. However, as binding partners of many protein IDRs are unknown, these structural transitions are difficult to verify and often are poorly understood. In this study we describe a method to identify IDRs that are likely to undergo helical transitions upon binding. This method combines bioinformatics analyses followed by circular dichroism spectroscopy to monitor 2,2,2-trifluoroethanol (TFE)-induced changes in secondary structure content of these IDRs. Our results demonstrate that there is no significant change in the helicity of IDRs that are not predicted to fold upon binding. IDRs that are predicted to fold fall into two groups: one group does not become helical in the presence of TFE and includes examples of IDRs that form β-strands upon binding, while the other group becomes more helical and includes examples that are known to fold into helices upon binding. Therefore, we propose that bioinformatics analyses combined with experimental evaluation using TFE may provide a general method to identify IDRs that undergo binding-induced disorder-to-helix transitions. PMID:27179590

  3. Solvation thermodynamics of amino acid side chains on a short peptide backbone

    SciTech Connect

    Hajari, Timir; Vegt, Nico F. A. van der

    2015-04-14

    The hydration process of side chain analogue molecules differs from that of the actual amino acid side chains in peptides and proteins owing to the effects of the peptide backbone on the aqueous solvent environment. A recent molecular simulation study has provided evidence that all nonpolar side chains, attached to a short peptide backbone, are considerably less hydrophobic than the free side chain analogue molecules. In contrast to this, the hydrophilicity of the polar side chains is hardly affected by the backbone. To analyze the origin of these observations, we here present a molecular simulation study on temperature dependent solvation free energies of nonpolar and polar side chains attached to a short peptide backbone. The estimated solvation entropies and enthalpies of the various amino acid side chains are compared with existing side chain analogue data. The solvation entropies and enthalpies of the polar side chains are negative, but in absolute magnitude smaller compared with the corresponding analogue data. The observed differences are large; however, owing to a nearly perfect enthalpy-entropy compensation, the solvation free energies of polar side chains remain largely unaffected by the peptide backbone. We find that a similar compensation does not apply to the nonpolar side chains; while the backbone greatly reduces the unfavorable solvation entropies, the solvation enthalpies are either more favorable or only marginally affected. This results in a very small unfavorable free energy cost, or even free energy gain, of solvating the nonpolar side chains in strong contrast to solvation of small hydrophobic or nonpolar molecules in bulk water. The solvation free energies of nonpolar side chains have been furthermore decomposed into a repulsive cavity formation contribution and an attractive dispersion free energy contribution. We find that cavity formation next to the peptide backbone is entropically favored over formation of similar sized nonpolar side

  4. Long-range conformational transition of a photoswitchable allosteric protein: molecular dynamics simulation study.

    PubMed

    Buchenberg, Sebastian; Knecht, Volker; Walser, Reto; Hamm, Peter; Stock, Gerhard

    2014-11-26

    A local perturbation of a protein may lead to functional changes at some distal site. An example is the PDZ2 domain of human tyrosine phosphatase 1E, which shows an allosteric transition upon binding to a peptide ligand. Recently Buchli et al. presented a time-resolved study of this transition by covalently linking an azobenzene photoswitch across the binding groove and using a femtosecond laser pulse that triggers the cis-trans photoisomerization of azobenzene. To aid the interpretation of these experiments, in this work seven microsecond runs of all-atom molecular dynamics simulations each for the wild-type PDZ2 in the ligand-bound and -free state, as well as the photoswitchable protein (PDZ2S) in the cis and trans states of the photoswitch, in explicit water were conducted. First the theoretical model is validated by recalculating the available NMR data from the simulations. By comparing the results for PDZ2 and PDZ2S, it is analyzed to what extent the photoswitch indeed mimics the free-bound transition. A detailed description of the conformational rearrangement following the cis-trans photoisomerization of PDZ2S reveals a series of photoinduced structural changes that propagate from the anchor residues of the photoswitch via intermediate secondary structure segments to the C-terminus of PDZ2S. The changes of the conformational distribution of the C-terminal region is considered as the distal response of the isolated allosteric protein. PMID:25365469

  5. Histidine-Directed Arylation/Alkenylation of Backbone N-H Bonds Mediated by Copper(II).

    PubMed

    Ohata, Jun; Minus, Matthew B; Abernathy, Morgan E; Ball, Zachary T

    2016-06-22

    Chemical modification of proteins and peptides represents a challenge of reaction design as well as an important biological tool. In contrast to side-chain modification, synthetic methods to alter backbone structure are extremely limited. In this communication, copper-mediated backbone N-alkenylation or N-arylation of peptides and proteins by direct modification of natural sequences is described. Histidine residues direct oxidative coupling of boronic acids at the backbone NH of a neighboring amino acid. The mild reaction conditions in common physiological buffers, at ambient temperature, are compatible with proteins and biological systems. This simple reaction demonstrates the potential for directed reactions in complex systems to allow modification of N-H bonds that directly affect polypeptide structure, stability, and function. PMID:27249339

  6. Extracting the Information Backbone in Online System

    PubMed Central

    Zhang, Qian-Ming; Zeng, An; Shang, Ming-Sheng

    2013-01-01

    Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity) of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such “less can be more” feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency. PMID:23690946

  7. Toward future IP optical backbone networks

    NASA Astrophysics Data System (ADS)

    Urushidani, Shigeo

    2005-11-01

    The rapid and aggressive penetration of broadband access services such as fiber to the home (FTTH) has been accelerating the increase in IP traffic volume and new networking technologies are required in order to accommodate future traffic in a cost-effective manner. This paper overviews the advanced IP optical network architecture and technologies for very-large-scale IP backbone networks. These technologies are the key to accommodate the huge volumes of IP traffic expected and control network resources in an effective and dynamic manner. We describe advanced IP optical networking technologies which accommodate multiple service networks using multi-instance technologies, and enable multi-layer traffic engineering using virtual network topology technologies. The migration scenario is described from the existing networks to GMPLS networks; reference is made to the advanced Path Computation Element (PCE) which enables multi-layer traffic engineering and MPLS/GMPLS migration. New network concepts such as Layer 1 Virtual Private Network (L1VPN) and GMPLS interoperability issues, which are being discussed in IETF, are also described.

  8. Extracting the information backbone in online system.

    PubMed

    Zhang, Qian-Ming; Zeng, An; Shang, Ming-Sheng

    2013-01-01

    Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity) of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such "less can be more" feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency. PMID:23690946

  9. A New Secondary Structure Assignment Algorithm Using Cα Backbone Fragments.

    PubMed

    Cao, Chen; Wang, Guishen; Liu, An; Xu, Shutan; Wang, Lincong; Zou, Shuxue

    2016-01-01

    The assignment of secondary structure elements in proteins is a key step in the analysis of their structures and functions. We have developed an algorithm, SACF (secondary structure assignment based on Cα fragments), for secondary structure element (SSE) assignment based on the alignment of Cα backbone fragments with central poses derived by clustering known SSE fragments. The assignment algorithm consists of three steps: First, the outlier fragments on known SSEs are detected. Next, the remaining fragments are clustered to obtain the central fragments for each cluster. Finally, the central fragments are used as a template to make assignments. Following a large-scale comparison of 11 secondary structure assignment methods, SACF, KAKSI and PROSS are found to have similar agreement with DSSP, while PCASSO agrees with DSSP best. SACF and PCASSO show preference to reducing residues in N and C cap regions, whereas KAKSI, P-SEA and SEGNO tend to add residues to the terminals when DSSP assignment is taken as standard. Moreover, our algorithm is able to assign subtle helices (310-helix, π-helix and left-handed helix) and make uniform assignments, as well as to detect rare SSEs in β-sheets or long helices as outlier fragments from other programs. The structural uniformity should be useful for protein structure classification and prediction, while outlier fragments underlie the structure-function relationship. PMID:26978354

  10. Bond distances in polypeptide backbones depend on the local conformation.

    PubMed

    Improta, Roberto; Vitagliano, Luigi; Esposito, Luciana

    2015-06-01

    By combining quantum-mechanical analysis of small model peptides and statistical surveys of high-resolution protein structures, a systematic conformational dependence of bond lengths in polypeptide backbones has been unveiled which involves both the peptide bond (C-O and C-N) and those bonds centred on the C(α) atom. All of these bond lengths indeed display a systematic variability in the ψ angle according to both calculations and surveys of protein structures. The overall agreement between the computed and the statistical data suggests that these trends are essentially driven by local effects. The dependence of C(α) distances on ψ is governed by interactions between the σ system of the C(α) moiety and the C-O π system of the peptide bond. Maximum and minimum values for each bond distance are found for conformations with the specific bond perpendicular and parallel to the adjacent CONH peptide plane, respectively. On the other hand, the variability of the C-O and C-N distances is related to the strength of the interactions between the lone pair of the N atom and the C-O π* system, which is modulated by the ψ angle. The C-O and C-N distances are related but their trends are not strictly connected to peptide-bond planarity, although a correlation amongst all of these parameters is expected on the basis of the classical resonance model. PMID:26057667

  11. A New Secondary Structure Assignment Algorithm Using Cα Backbone Fragments

    PubMed Central

    Cao, Chen; Wang, Guishen; Liu, An; Xu, Shutan; Wang, Lincong; Zou, Shuxue

    2016-01-01

    The assignment of secondary structure elements in proteins is a key step in the analysis of their structures and functions. We have developed an algorithm, SACF (secondary structure assignment based on Cα fragments), for secondary structure element (SSE) assignment based on the alignment of Cα backbone fragments with central poses derived by clustering known SSE fragments. The assignment algorithm consists of three steps: First, the outlier fragments on known SSEs are detected. Next, the remaining fragments are clustered to obtain the central fragments for each cluster. Finally, the central fragments are used as a template to make assignments. Following a large-scale comparison of 11 secondary structure assignment methods, SACF, KAKSI and PROSS are found to have similar agreement with DSSP, while PCASSO agrees with DSSP best. SACF and PCASSO show preference to reducing residues in N and C cap regions, whereas KAKSI, P-SEA and SEGNO tend to add residues to the terminals when DSSP assignment is taken as standard. Moreover, our algorithm is able to assign subtle helices (310-helix, π-helix and left-handed helix) and make uniform assignments, as well as to detect rare SSEs in β-sheets or long helices as outlier fragments from other programs. The structural uniformity should be useful for protein structure classification and prediction, while outlier fragments underlie the structure–function relationship. PMID:26978354

  12. F-box proteins: Keeping the epithelial-to-mesenchymal transition (EMT) in check.

    PubMed

    Díaz, Víctor M; de Herreros, Antonio García

    2016-02-01

    F-box proteins are the key recognition subunit of multimeric E3 ubiquitin ligase complexes that participate in the proteasome degradation of specific substrates. In the last years, a discrete number of F-box proteins have been shown to regulate the epithelial-to-mesenchymal transition (EMT), a process defined by a rapid change of cell phenotype, the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Specific EMT transcription factors (EMT-TFs), such as Snail, Slug, Twist and Zeb, control EMT induction both during development and in cancer. These EMT-TFs are short-lived proteins that are targeted to the proteasome system by specific F-box proteins, keeping them at low levels. F-box proteins also indirectly regulate the EMT process by controlling EMT inducers, such as Notch, c-Myc or mTOR. Here we summarize the role that these F-box proteins (Fbxw1, Fbxw7, Fbxl14, Fbxl5, Fbxo11 and Fbxo45) play in controlling EMT during development and cancer progression, a process dependent on post-translational modifications that govern their interaction with target proteins. PMID:26506454

  13. Transition fibre protein FBF1 is required for the ciliary entry of assembled intraflagellar transport complexes.

    PubMed

    Wei, Qing; Xu, Qingwen; Zhang, Yuxia; Li, Yujie; Zhang, Qing; Hu, Zeng; Harris, Peter C; Torres, Vicente E; Ling, Kun; Hu, Jinghua

    2013-01-01

    Sensory organelle cilia have critical roles in mammalian embryonic development and tissue homeostasis. Intraflagellar transport (IFT) machinery is required for the assembly and maintenance of cilia. Yet, how this large complex passes through the size-dependent barrier at the ciliary base remains enigmatic. Here we report that FBF1, a highly conserved transition fibre protein, is required for the ciliary import of assembled IFT particles at the ciliary base. We cloned dyf-19, the Caenorhabditis elegans homologue of human FBF1, in a whole-genome screen for ciliogenesis mutants. DYF-19 localizes specifically to transition fibres and interacts directly with the IFT-B component DYF-11/IFT54. Although not a structural component of transition fibres, DYF-19 is required for the transit of assembled IFT particles through the ciliary base. Furthermore, we found that human FBF1 shares conserved localization and function with its worm counterpart. We conclude that FBF1 is a key functional transition fibre component that actively facilitates the ciliary entry of assembled IFT machinery. PMID:24231678

  14. Data on structural transitions in domains of hordeivirus TGB1 protein forming ribonucleoprotein complex.

    PubMed

    Makarov, Valentin V; Makarova, Svetlana S; Kalinina, Natalia O

    2016-09-01

    This data article is related to the research article entitled "in vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes" (Makarov et al., 2015 [1]), demonstrating that upon incubation with viral RNA the poa semilatent hordeivirus (PSLV) TGB1 protein (the movement 63 K protein encoded by the first gene of the triple gene block) in vitro forms RNP structures resembling filamentous virus-like particles and its internal domain (ID) performs a major structural role in this process. This article reports the additional results on the structural lability of ID and the structural transitions in the C-terminal NTPase/helicase domain (HELD) induced by interaction with tRNA and phosphorylation. PMID:27331098

  15. Phase transition of spindle-associated protein regulate spindle apparatus assembly.

    PubMed

    Jiang, Hao; Wang, Shusheng; Huang, Yuejia; He, Xiaonan; Cui, Honggang; Zhu, Xueliang; Zheng, Yixian

    2015-09-24

    Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks. PMID:26388440

  16. Structural transitions in the intrinsically disordered Parkinson's protein alpha-synuclein

    NASA Astrophysics Data System (ADS)

    Eliezer, David

    2013-03-01

    The protein alpha-synuclein is genetically and histopathologically associated with familial and sporadic Parkinson's disease. Although considered to belong to the category of intrinsically disordered proteins for well over a decade, recent reports have suggested that synuclein may actually exist predominantly in a native, well-structured, tetrameric form. Experiments using in-cell NMR, which bypass potential structural perturbations caused by purification protocols, conclusively demonstrate that recombinant synuclein is in fact highly disordered and monomeric. In the presence of membranes, however, the protein undergoes a coil-to-helix transition to adopt several highly helical conformations, which are proposed to mediate both its normal function and its membrane-induced aggregation into amyloid fibrils. Supported by NIH grant R37AG019391

  17. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  18. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.

    PubMed

    Patel, Avinash; Lee, Hyun O; Jawerth, Louise; Maharana, Shovamayee; Jahnel, Marcus; Hein, Marco Y; Stoynov, Stoyno; Mahamid, Julia; Saha, Shambaditya; Franzmann, Titus M; Pozniakovski, Andrej; Poser, Ina; Maghelli, Nicola; Royer, Loic A; Weigert, Martin; Myers, Eugene W; Grill, Stephan; Drechsel, David; Hyman, Anthony A; Alberti, Simon

    2015-08-27

    Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases. PMID:26317470

  19. Altered expression of epithelial-to-mesenchymal transition proteins in extraprostatic prostate cancer

    PubMed Central

    Verrill, Clare; Cerundolo, Lucia; Mckee, Chad; White, Michael; Kartsonaki, Christiana; Fryer, Eve; Morris, Emma; Brewster, Simon; Ratnayaka, Indrika; Marsden, Luke; Lilja, Hans; Muschel, Ruth; Lu, Xin; Hamdy, Freddie; Bryant, Richard J.

    2016-01-01

    Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten−/− model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models. PMID:26701730

  20. Developmental Staging of Maize Microspores Reveals a Transition in Developing Microspore Proteins 1

    PubMed Central

    Bedinger, Patricia A.; Edgerton, Michael D.

    1990-01-01

    A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady-state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16667300

  1. Cataloging proteins putatively secreted during the biotrophy-necrotrophy transition of the anthracnose pathogen Colletotrichum truncatum.

    PubMed

    Bhadauria, Vijai; Banniza, Sabine; Vandenberg, Albert; Selvaraj, Gopalan; Wei, Yangdou

    2011-10-01

    Hemibiotrophic phytopathogenic fungi cause devastating diseases in agronomically important crops. These fungal pathogens exploit a stealth bi-phasic infection strategy to colonize host plants. Their morphological and nutritional transition from biotrophy (characterized by voluminous intracellular primary hyphae) to necrotrophy (characterized by thin secondary hyphae) known as the biotrophy-necrotrophy switch (hemibiotrophy) is critical in symptom and disease development. To establish successful hemibiotrophic parasitism, pathogens likely secrete suites of proteins at the switch that constitute the biotrophy-necrotrophy switch secretome. To catalogue such proteins, a directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the switch of Colletotrichum truncatum, and 5000 expressed sequence tags (ESTs) were generated. Four potential groups (hydrolytic enzymes, cell envelope-associated proteins [CEAPs], candidate effectors and proteins with diverse functions) were identified from pathogen-derived ESTs. Expression profiling of transcripts encoding CEAPs and candidate effectors in an infection time-course revealed that the majority of these transcripts were expressed or induced during the necrotrophic phase and repressed during the biotrophic phase of in planta colonization, indicating the massive accumulation of proteins at the switch. Taken together, our data suggest that the hemibiotrophic mode of fungal proliferation entails complex interactions of a pathogen with its host wherein the pathogen requires live host cells prior to switching to the necrotrophic phase. The microbial proteins employed during pathogenesis are likely to have defined roles at specific stages of pathogenesis. PMID:21897125

  2. Structural basis of conformational transitions in the active site and 80′s loop in the FK506-binding protein FKBP12

    PubMed Central

    Mustafi, Sourajit M.; Brecher, Matthew; Zhang, Jing; Li, Hongmin; Lemaster, David M.; Hernández, Griselda

    2014-01-01

    The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80′s loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80′s loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans–cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80′s loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80′s loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50′s loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue. PMID:24405377

  3. Backbone and side chain NMR assignments for the ribosome assembly factor Nop6 from Saccharomyces cerevisiae.

    PubMed

    Wurm, Jan Philip; Lioutikov, Anatoli; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2014-10-01

    The Saccharomyces cerevisiae Nop6 protein is involved in the maturation of the small ribosomal subunit. It contains a central RNA binding domain and a predicted C-terminal coiled-coil domain. Here we report the almost complete (>90%) (1)H,(13)C,(15)N backbone and side chain NMR assignment of a 15 kDa Nop6 construct comprising the RNA binding and coiled-coil domains. PMID:23921755

  4. Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

    SciTech Connect

    Ho, Meng-Chiao; Sturm, Matthew B.; Almo, Steven C.; Schramm, Vern L.

    2010-01-12

    Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple {pi}-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the {pi}-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H{sub 2}O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.

  5. Radiation safety system (RSS) backbones: Design, engineering, fabrication and installation

    SciTech Connect

    Wilmarth, J.E.; Sturrock, J.C.; Gallegos, F.R.

    1998-12-01

    The Radiation Safety System (RSS) Backbones are part of an electrical/electronic/mechanical system insuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS Backbones control the safety fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low energy beam transport. The Backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the Backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two Linac Backbone segments and experimental area segments form a continuous cable plant over 3,500 feet from beam plugs to the tip on the longest tail. The Backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely.

  6. Radiation Safety System (RSS) backbones: Design, engineering, fabrication, and installation

    SciTech Connect

    Wilmarth, J. E.; Sturrock, J. C.; Gallegos, F. R.

    1998-12-10

    The Radiation Safety System (RSS) backbones are part of an electrical/electronic/mechanical system ensuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS backbones control the safety-fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low-energy beam transport. The backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two linac backbone segments and the experimental area segments form a continuous cable plant over 3500 feet from the beam plugs to the tip on the longest tail. The backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely.

  7. Radiation Safety System (RSS) backbones: Design, engineering, fabrication, and installation

    NASA Astrophysics Data System (ADS)

    Wilmarth, J. E.; Sturrock, J. C.; Gallegos, F. R.

    1998-12-01

    The Radiation Safety System (RSS) backbones are part of an electrical/electronic/mechanical system ensuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS backbones control the safety-fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low-energy beam transport. The backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two linac backbone segments and the experimental area segments form a continuous cable plant over 3500 feet from the beam plugs to the tip on the longest tail. The backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely.

  8. Radiation Safety System (RSS) backbones: Design, engineering, fabrication, and installation

    SciTech Connect

    Wilmarth, J.E.; Sturrock, J.C.; Gallegos, F.R.

    1998-12-01

    The Radiation Safety System (RSS) backbones are part of an electrical/electronic/mechanical system ensuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS backbones control the safety-fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low-energy beam transport. The backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two linac backbone segments and the experimental area segments form a continuous cable plant over 3500 feet from the beam plugs to the tip on the longest tail. The backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely. {copyright} {ital 1998 American Institute of Physics.}

  9. Growth Kinetics of Intracellular RNA/Protein Droplets: Signature of a Liquid-Liquid Phase Transition?

    NASA Astrophysics Data System (ADS)

    Berry, Joel; Weber, Stephanie C.; Vaidya, Nilesh; Zhu, Lian; Haataja, Mikko; Brangwynne, Clifford P.

    2015-03-01

    Nonmembrane-bound organelles are functional, dynamic assemblies of RNA and/or protein that can self-assemble and disassemble within the cytoplasm or nucleoplasm. The possibility that underlying intracellular phase transitions may drive and mediate the morphological evolution of some membrane-less organelles has been supported by several recent studies. In this talk, results from a collaborative experimental-theoretical study of the growth and dissolution kinetics of nucleoli and extranucleolar droplets (ENDs) in C. elegans embryos will be presented. We have employed Flory-Huggins solution theory, reaction-diffusion kinetics, and quantitative statistical dynamic scaling analysis to characterize the specific growth mechanisms at work. Our findings indicate that both in vivo and in vitro droplet scaling and growth kinetics are consistent with those resulting from an equilibrium liquid-liquid phase transition mediated by passive nonequilibrium growth mechanisms - simultaneous Brownian coalescence and Ostwald ripening. This supports a view in which cells can employ phase transitions to drive structural organization, while utilizing active processes, such as local transcriptional activity, to fine tune the kinetics of these phase transitions in response to given conditions.

  10. Alteration of protein synthesis in Saccharomyces cerevisiae caused by transition metals

    SciTech Connect

    Chang, E.C.; Willsky, G.R.; Kosman, D.J.

    1986-05-01

    The effects of transition metals and metal oxides (Co/sup 2 +/, Cu/sup 2 +/, Ni/sup 2 +/, Zn/sup 2 +/, arsenite (AsO/sub 2//sup -/), arsenate (AsO/sub 4//sup 3 -/) and vanadate (VO/sub 4//sup 3 -/)) on protein synthesis and cellular thermotolerance in Saccharomyces cerevisiae have been examined. Arsenate and vanadate differ in that they are known to be physiologic phosphate analogues, e.g. in yeast, arsenate is a competitive inhibitor of phosphate transport and is incorporated into phosphoinositide, while vanadate inhibits the formation of various phosphoproteins and phosphoprotein intermediates. Arsenite and arsenate at concentrations above 0.5 mM and 5 mM, respectively, in minimal glucose media inhibited growth. These two oxides at 1 mM induced 4 of the 6 major heat shock proteins (Hsps): 105, 78, 74 and 36 Kd. The 36 Kd protein as not induced by arsenite at 0.1 mM. Vanadate induced no known Hsp but did induce one polypeptide of 155 Kd. These metal-induced proteins did not confer cellular thermotolerance. No protein induction was observed after 45 min preincubation with 0.5 mM divalent Co, Cu, Ni or Zn despite the fact that as with the metal oxides a significant inhibition of growth and over-all protein synthesis was caused by Co/sup 2 +/, Cu/sup 2 +/ and Ni/sup 2 +/ at that concentration. Thus, among this group of metals the induction of specific proteins appears to be unique to those species which mimic the phosphate group. Stress which alters the growth to cells does not always trigger a specific alteration of protein synthesis.

  11. Generic transition hierarchies of lattice HP protein adsorption: A Wang-Landau study

    NASA Astrophysics Data System (ADS)

    Li, Ying Wai; Landau, D. P.; Wüst, T.

    2012-02-01

    We have applied Wang-Landau sampling with appropriate trial movesootnotetextT. W"ust and D. P. Landau, Phy. Rev. Lett. 102, 178101 (2009). to investigate the thermodynamics and structural properties of the HP lattice protein modelootnotetextK. A. Dill, Biochemistry 24, 1501 (1985). interacting with an attractive substrate. The conformational ``phase transitions'' of several benchmark HP sequences have been identified by a comprehensive canonical analysis of the specific heat and structural observables, e. g. radius of gyration and thermal derivatives of number of surface contacts. Three major ``transitions'': adsorption, hydrophobic core formation, and ``flattening'' of adsorbed structures, are observed. Depending on the surface attractive strength relative to the intra-protein attraction among the H monomers, these processes take place in a different order upon cooling. We identify a small number of generic categories that are sufficient to classify the folding hierarchies for different HP chains consisting of assorted sequences and chain lengths, regardless of the monomer type that the surface attracts. We thus believe that this classification scheme is generally applicable to lattice protein adsorption problems.

  12. Smooth Functional Transition along a Mutational Pathway with an Abrupt Protein Fold Switch

    PubMed Central

    Holzgräfe, Christian; Wallin, Stefan

    2014-01-01

    Recent protein design experiments have demonstrated that proteins can migrate between folds through the accumulation of substitution mutations without visiting disordered or nonfunctional points in sequence space. To explore the biophysical mechanism underlying such transitions we use a three-letter continuous protein model with seven atoms per amino acid to provide realistic sequence-structure and sequence-function mappings through explicit simulation of the folding and interaction of model sequences. We start from two 16-amino-acid sequences folding into an α-helix and a β-hairpin, respectively, each of which has a preferred binding partner with 35 amino acids. We identify a mutational pathway between the two folds, which features a sharp fold switch. By contrast, we find that the transition in function is smooth. Moreover, the switch in preferred binding partner does not coincide with the fold switch. Discovery of new folds in evolution might therefore be facilitated by following fitness slopes in sequence space underpinned by binding-induced conformational switching. PMID:25185557

  13. Controlling the Protein Dynamical Transition with Sugar-Based Bioprotectant Matrices: A Neutron Scattering Study

    PubMed Central

    Cornicchi, E.; Marconi, M.; Onori, G.; Paciaroni, A.

    2006-01-01

    Through elastic neutron scattering we measured the mean-square displacements of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temperature and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temperature range. The dry sample shows an onset of anharmonicity at ∼100 K, which can be attributed to the activation of methyl group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temperature range. In the hydrated samples an additional and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temperature Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by ∼60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response versus T and the thermal stability in glucose-water bioprotectant matrices is proposed. PMID:16617083

  14. Shifting transition states in the unfolding of a large ankyrin repeat protein

    PubMed Central

    Werbeck, Nicolas D.; Rowling, Pamela J. E.; Chellamuthu, Vasuki R.; Itzhaki, Laura S.

    2008-01-01

    The 33-amino-acid ankyrin motif comprises a β-turn followed by two anti-parallel α-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a “seed” and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it. PMID:18632570

  15. The Onset of Collective Structural Vibrations at the Protein Dynamical Transition

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine A.; Deng, Yanting; Michki, Nigel S.; Snell, Edward H.; Markelz, Andrea G.

    X-ray, neutron scattering and terahertz measurements found a rapid increase in dynamics of critically hydrated proteins above 220 K, termed the protein dynamical transition. Protein function ceases below the DT. It has been suggested that protein dynamics is slaved to the solvent and the DT originates from thermally activated solvent motions. Since previous measurements did not distinguish local diffusive and librational motions from long-range collective vibrations of proteins, it has not been determined how long-range motions are affected by the DT. Using a recently developed technique, crystal anisotropy terahertz microscopy we directly measured the long-range motions for lysozyme and examined the temperature dependence in the 180-290 K range. We find that the distinct intramolecular vibrations do not follow the expected phonon-like behavior of solid state systems where the vibrational bands sharpen and blue shift with decreasing temperature, rather decrease in intensity as the DT is approached and disappear below the DT. This suggests the surrounding solvent below the DT acts as a frozen cage preventing long-range motions.

  16. Modeling protein conformational transitions by a combination of coarse-grained normal mode analysis and robotics-inspired methods

    PubMed Central

    2013-01-01

    Background Obtaining atomic-scale information about large-amplitude conformational transitions in proteins is a challenging problem for both experimental and computational methods. Such information is, however, important for understanding the mechanisms of interaction of many proteins. Methods This paper presents a computationally efficient approach, combining methods originating from robotics and computational biophysics, to model protein conformational transitions. The ability of normal mode analysis to predict directions of collective, large-amplitude motions is applied to bias the conformational exploration performed by a motion planning algorithm. To reduce the dimension of the problem, normal modes are computed for a coarse-grained elastic network model built on short fragments of three residues. Nevertheless, the validity of intermediate conformations is checked using the all-atom model, which is accurately reconstructed from the coarse-grained one using closed-form inverse kinematics. Results Tests on a set of ten proteins demonstrate the ability of the method to model conformational transitions of proteins within a few hours of computing time on a single processor. These results also show that the computing time scales linearly with the protein size, independently of the protein topology. Further experiments on adenylate kinase show that main features of the transition between the open and closed conformations of this protein are well captured in the computed path. Conclusions The proposed method enables the simulation of large-amplitude conformational transitions in proteins using very few computational resources. The resulting paths are a first approximation that can directly provide important information on the molecular mechanisms involved in the conformational transition. This approximation can be subsequently refined and analyzed using state-of-the-art energy models and molecular modeling methods. PMID:24564964

  17. Constructing optimal backbone segments for joining fixed DNA base pairs.

    PubMed Central

    Mazur, J; Jernigan, R L; Sarai, A

    1996-01-01

    A method is presented to link a sequence of space-fixed base pairs by the sugar-phosphate segments of single nucleotides and to evaluate the effects in the backbone caused by this positioning of the bases. The entire computational unit comprises several nucleotides that are energy-minimized, subject to constraints imposed by the sugar-phosphate backbone segments being anchored to space-fixed base pairs. The minimization schemes are based on two stages, a conjugate gradient method followed by a Newton-Raphson algorithm. Because our purpose is to examine the response, or relaxation, of an artificially stressed backbone, it is essential to be able to obtain, as closely as possible, a lowest minimum energy conformation of the backbone segment in conformational space. For this purpose, an algorithm is developed that leads to the generation of an assembly of many local energy minima. From these sets of local minima, one conformation corresponding to the one with the lowest minimum is then selected and designated to represent the backbone segment at its minimum. The effective electrostatic potential of mean force is expressed in terms of adjustable parameters that incorporate solvent screening action in the Coulombic interactions between charged backbone atoms; these parameters are adjusted to obtain the best fit of the nearest-neighbor phosphorous atoms in an x-ray structure. PMID:8874023

  18. Large-scale measurement and modeling of backbone Internet traffic

    NASA Astrophysics Data System (ADS)

    Roughan, Matthew; Gottlieb, Joel

    2002-07-01

    There is a brewing controversy in the traffic modeling community concerning how to model backbone traffic. The fundamental work on self-similarity in data traffic appears to be contradicted by recent findings that suggest that backbone traffic is smooth. The traffic analysis work to date has focused on high-quality but limited-scope packet trace measurements; this limits its applicability to high-speed backbone traffic. This paper uses more than one year's worth of SNMP traffic data covering an entire Tier 1 ISP backbone to address the question of how backbone network traffic should be modeled. Although the limitations of SNMP measurements do not permit us to comment on the fine timescale behavior of the traffic, careful analysis of the data suggests that irrespective of the variation at fine timescales, we can construct a simple traffic model that captures key features of the observed traffic. Furthermore, the model's parameters are measurable using existing network infrastructure, making this model practical in a present-day operational network. In addition to its practicality, the model verifies basic statistical multiplexing results, and thus sheds deep insight into how smooth backbone traffic really is.

  19. Slow transition between two beta-strand registers is dictated by protein unfolding.

    PubMed

    Evans, Matthew R; Gardner, Kevin H

    2009-08-19

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous, basic helix-loop-helix Period/ARNT/Single-minded protein that forms dimeric transcriptional regulator complexes with other bHLH-PAS proteins to regulate various biological pathways. Intriguingly, the introduction of a single point mutation into the C-terminal PAS-B domain resulted in a protein that can simultaneously exist in two distinct conformations. The difference between these two structures is a +3 slip and inversion of a central Ibeta-strand and an accompanying N448-P449 peptide bond isomerization in the preceding HI loop. Previous studies have indicated these two forms of Y456T interconvert on the approximate time scale of tens of minutes, allowing these two conformations to be separated by ion exchange chromatography. Here, we use time-resolved solution NMR spectroscopy to quantitatively characterize this rate and its temperature dependence, providing information into the transition state. When compared with fluorescence measurements of protein unfolding rates, we find data that suggest a linkage between interconversion and unfolding based on comparable temperature dependence and corresponding energetics of these processes. Notably, the N448-P449 peptide bond also plays a critical role for the interconversion between states, with a mutant unable to adopt a cis configuration at this bond (P449A/Y456T) being kinetically trapped under nondenaturing conditions. Taken together, these data provide information about a rare equilibrium model system for beta-strand slippage. PMID:19722642

  20. Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation.

    PubMed

    Qvit, Nir; Kornfeld, Opher S

    2016-01-01

    Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency. Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position. We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites' viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a

  1. Dephosphorylation of the Rieske iron-sulfur protein after induction of the mitochondrial permeability transition

    SciTech Connect

    He Lihua; Lemasters, John J. . E-mail: lemaster@med.unc.edu

    2005-09-02

    In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl{sub 2}, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS-PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron-sulfur protein (RISP) of ubiquinol-cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS-PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol-cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.

  2. Deciphering conformational transitions of proteins by small angle X-ray scattering and normal mode analysis.

    PubMed

    Panjkovich, Alejandro; Svergun, Dmitri I

    2016-02-17

    Structural flexibility and conformational rearrangements are often related to important functions of biological macromolecules, but the experimental characterization of such transitions with high-resolution techniques is challenging. At a lower resolution, small angle X-ray scattering (SAXS) can be used to obtain information on biomolecular shapes and transitions in solution. Here, we present SREFLEX, a hybrid modeling approach that uses normal mode analysis (NMA) to explore the conformational space of high-resolution models and refine the structure guided by the agreement with the experimental SAXS data. The method starts from a given conformation of the protein (which does not agree with the SAXS data). The structure is partitioned into pseudo-domains either using structural classification databases or automatically from the protein dynamics as predicted by the NMA. The algorithm proceeds hierarchically employing NMA to first probe large rearrangements and progresses into smaller and more localized movements. At the large rearrangements stage the pseudo-domains stay as rigid bodies allowing one to avoid structural disruptions inherent to the earlier NMA-based algorithms. To validate the approach, we compiled a representative benchmark set of 88 conformational states known experimentally at high resolution. The performance of the algorithm is demonstrated in the simulated data on the benchmark set and also in a number of experimental examples. SREFLEX is included into the ATSAS program package freely available to the academic users, both for download and in the on-line mode. PMID:26611321

  3. Fighting Cancer with Transition Metal Complexes: From Naked DNA to Protein and Chromatin Targeting Strategies.

    PubMed

    Palermo, Giulia; Magistrato, Alessandra; Riedel, Tina; von Erlach, Thibaud; Davey, Curt A; Dyson, Paul J; Rothlisberger, Ursula

    2016-06-20

    Many transition metal complexes have unique physicochemical properties that can be efficiently exploited in medicinal chemistry for cancer treatment. Traditionally, double-stranded DNA has been assumed to be the main binding target; however, recent studies have shown that nucleosomal DNA as well as proteins can act as dominant molecular binding partners. This has raised new questions about the molecular determinants that govern DNA versus protein binding selectivity, and has offered new ways to rationalize their biological activity and possible side effects. To address these questions, molecular simulations at an atomistic level of detail have been used to complement, support, and rationalize experimental data. Herein we review some relevant studies-focused on platinum and ruthenium compounds-to illustrate the power of state-of-the-art molecular simulation techniques and to demonstrate how the interplay between molecular simulations and experiments can make important contributions to elucidating the target preferences of some promising transition metal anticancer agents. This contribution aims at providing relevant information that may help in the rational design of novel drug-discovery strategies. PMID:26634638

  4. Epithelial-Mesenchymal Transition Protein Expression in Basal Cell Adenomas and Basal Cell Adenocarcinomas.

    PubMed

    Tesdahl, Brennan A; Wilson, Thomas C; Hoffman, Henry T; Robinson, Robert A

    2016-06-01

    Basal cell adenomas and basal cell adenocarcinomas show marked histomorphologic similarity and are separated microscopically primarily by the invasive characteristics of the adenocarcinomas. We wished to explore potential differences in the expression of epithelial-mesenchymal transition associated proteins in these two tumor types. A tissue microarray was constructed utilizing 29 basal cell adenomas and 16 basal cell adenocarcinomas. Immunohistochemical expression of E-cadherin, beta-catenin, Twist 1 and vimentin were investigated. Both tumors expressed all proteins in a relatively similar manner. Nuclear beta-catenin was essentially limited to the abluminal cell populations in both tumor types. E-cadherin was limited largely to luminal locations but was more prevalent in the adenocarcinomas as compared to the adenomas. Primarily abluminal expression for vimentin was seen, sometimes present in an apical dot-like pattern. Distinct populations of cellular expression of these four markers of epithelial mesenchymal transition were present but were similar in locations in both tumors with no patterns discerned to separate basal cell adenoma from basal cell adenocarcinoma. Given these findings, the mechanisms by which basal cell adenocarcinoma is able to invade while its counterpart, basal cell adenoma can not, may be more complex than in other tumor types. PMID:26442856

  5. Backbone and sidechain 1H, 15N and 13C assignments of the KSR1 CA1 domain

    PubMed Central

    Koveal, Dorothy; Pinheiro, Anderson S.; Peti, Wolfgang; Page, Rebecca

    2014-01-01

    The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [13C, 15N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain. PMID:20737253

  6. Short communication: Proteins from circulating exosomes represent metabolic state in transition dairy cows.

    PubMed

    Crookenden, M A; Walker, C G; Peiris, H; Koh, Y; Heiser, A; Loor, J J; Moyes, K M; Murray, A; Dukkipati, V S R; Kay, J K; Meier, S; Roche, J R; Mitchell, M D

    2016-09-01

    provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state. PMID:27320663

  7. Detection of gene annotations and protein-protein interaction associated disorders through transitive relationships between integrated annotations

    PubMed Central

    2015-01-01

    Background Increasingly high amounts of heterogeneous and valuable controlled biomolecular annotations are available, but far from exhaustive and scattered in many databases. Several annotation integration and prediction approaches have been proposed, but these issues are still unsolved. We previously created a Genomic and Proteomic Knowledge Base (GPKB) that efficiently integrates many distributed biomolecular annotation and interaction data of several organisms, including 32,956,102 gene annotations, 273,522,470 protein annotations and 277,095 protein-protein interactions (PPIs). Results By comprehensively leveraging transitive relationships defined by the numerous association data integrated in GPKB, we developed a software procedure that effectively detects and supplement consistent biomolecular annotations not present in the integrated sources. According to some defined logic rules, it does so only when the semantic type of data and of their relationships, as well as the cardinality of the relationships, allow identifying molecular biology compliant annotations. Thanks to controlled consistency and quality enforced on data integrated in GPKB, and to the procedures used to avoid error propagation during their automatic processing, we could reliably identify many annotations, which we integrated in GPKB. They comprise 3,144 gene to pathway and 21,942 gene to biological function annotations of many organisms, and 1,027 candidate associations between 317 genetic disorders and 782 human PPIs. Overall estimated recall and precision of our approach were 90.56 % and 96.61 %, respectively. Co-functional evaluation of genes with known function showed high functional similarity between genes with new detected and known annotation to the same pathway; considering also the new detected gene functional annotations enhanced such functional similarity, which resembled the one existing between genes known to be annotated to the same pathway. Strong evidence was also found in

  8. Solvent effects in the helix-coil transition model can explain the unusual biophysics of intrinsically disordered proteins

    NASA Astrophysics Data System (ADS)

    Badasyan, Artem; Mamasakhlisov, Yevgeni Sh.; Podgornik, Rudolf; Parsegian, V. Adrian

    2015-07-01

    We analyze a model statistical description of the polypeptide chain helix-coil transition, where we take into account the specificity of its primary sequence, as quantified by the phase space volume ratio of the number of all accessible states to the number corresponding to a helical conformation. The resulting transition phase diagram is then juxtaposed with the unusual behavior of the secondary structures in Intrinsically Disordered Proteins (IDPs) and a number of similarities are observed, even if the protein folding is a more complex transition than the helix-coil transition. In fact, the deficit in bulky and hydrophobic amino acids observed in IDPs, translated into larger values of phase space volume, allows us to locate the region in parameter space of the helix-coil transition that would correspond to the secondary structure transformations that are intrinsic to conformational transitions in IDPs and that is characterized by a modified phase diagram when compared to globular proteins. Here, we argue how the nature of this modified phase diagram, obtained from a model of the helix-coil transition in a solvent, would illuminate the turned-out response of IDPs to the changes in the environment conditions that follow straightforwardly from the re-entrant (cold denaturation) branch in their folding phase diagram.

  9. Force-dependent switch in protein unfolding pathways and transition-state movements.

    PubMed

    Zhuravlev, Pavel I; Hinczewski, Michael; Chakrabarti, Shaon; Marqusee, Susan; Thirumalai, D

    2016-02-01

    Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, [Formula: see text], as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a [Formula: see text] plot. Similar observations were reported for other proteins for the unfolding rate [Formula: see text]. These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [Formula: see text] is increased from a low to a high value. We provide a unified theory demonstrating that if [Formula: see text] as a function of a perturbation (f or [Formula: see text]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in [Formula: see text] to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants. PMID:26818842

  10. Peptide Amphiphile Nanofibers with Conjugated Polydiacetylene Backbones in Their Core

    PubMed Central

    Hsu, Lorraine; Cvetanovich, Gregory L.; Stupp, Samuel I.

    2008-01-01

    The coupling of electronic and biological functionality through self-assembly is an interesting target in supramolecular chemistry. We report here on a set of diacetylene-derivatized peptide amphiphiles (PAs) that react to form conjugated polydiacetylene backbones following self-assembly into cylindrical nanofibers. The polymerization reaction yields highly conjugated backbones when the peptidic segment of the PAs has a linear, as opposed to a branched, architecture. Given the topotactic nature of the polymerization, these results suggest that a high degree of internal order exists in the supramolecular nanofibers formed by the linear PA. On the basis of microscopy, the formation of a polydiacetylene backbone to covalently connect the β-sheets that help form the fibers does not disrupt the fiber shape. Interestingly, we observe the appearance of a polydiacetylene (PDA) circular dichroism band at 547 nm in linear PA nanofibers suggesting the conjugated backbone in the core of the nanostructures is twisted. We believe this CD signal is due to chiral induction by the β-sheets, which are normally twisted in helical fashion. Heating and cooling shows simultaneous changes in β-sheet and conjugated backbone structure, indicating they are both correlated. At the same time, poor polymerization in nanofibers formed by branched PAs indicates that less internal order exists in these nanostructures and, as expected, then a circular dichroism signal is not observed for the conjugated backbone. The general variety of materials investigated here has the obvious potential to couple electronic properties and in vitro bioactivity. Furthermore, the polymerization of monomers in peptide amphiphile assemblies by a rigid conjugated backbone also leads to mechanical robustness and insolubility, two properties that may be important for the patterning of these materials at the cellular scale. PMID:18314978

  11. Alpha-helical stabilization by side chain shielding of backbone hydrogen bonds.

    PubMed

    García, Angel E; Sanbonmatsu, Kevin Y

    2002-03-01

    We study atomic models of the thermodynamics of the structural transition of peptides that form alpha-helices. The effect of sequence variation on alpha-helix formation for alanine-rich peptides, Ac-Ala21-methyl amide (A21) and Ac-A5 (AAARA)3A-methyl amide (Fs peptide), is investigated by atomic simulation studies of the thermodynamics of the helix-coil transition in explicit water. The simulations show that the guanidinium group in the Arg side chains in the Fs peptide interacts with the carbonyl group four amino acids upstream in the chain and desolvates backbone hydrogen bonds. This desolvation can be directly correlated with a higher probability of hydrogen bond formation. We find that Fs has higher helical content than A21 at all temperatures. A small modification in the amber force field reproduces the experimental helical content and helix-coil transition temperatures for the Fs peptide. PMID:11867710

  12. Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells.

    PubMed

    Prunotto, Marco; Compagnone, Alessandra; Bruschi, Maurizio; Candiano, Giovanni; Colombatto, Sebastiano; Bandino, Andrea; Petretto, Andrea; Moll, Solange; Bochaton-Piallat, Marie Luce; Gabbiani, Giulio; Dimuccio, Veronica; Parola, Maurizio; Citti, Lorenzo; Ghiggeri, Gianmarco

    2010-06-01

    Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis. PMID:20212449

  13. Mapping the structure and conformational movements of proteins with transition metal ion FRET

    PubMed Central

    Taraska, Justin W.; Puljung, Michael C.; Olivier, Nelson B.; Flynn, Galen E.; Zagotta, William N.

    2009-01-01

    SUMMARY Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here, we report that FRET between a small fluorescent dye and a nickel ion bound to a di-histidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET including the ability to measure the dynamics of close range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this ‘transition metal ion FRET’ approach along with x-ray crystallography to determine the structural changes of the gating-ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Binding of cAMP to the isolated carboxyl-terminal region of HCN2 caused a structural rearrangement involving a movement of the C-helix towards the β-roll of the cAMP-binding domain and a movement of the F′ helix of the C-linker, along with a stabilization of the secondary structure of the helices. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels. PMID:19525958

  14. Protein Induces Layer-by-Layer Exfoliation of Transition Metal Dichalcogenides.

    PubMed

    Guan, Guijian; Zhang, Shuangyuan; Liu, Shuhua; Cai, Yongqing; Low, Michelle; Teng, Choon Peng; Phang, In Yee; Cheng, Yuan; Duei, Koh Leng; Srinivasan, Bharathi Madurai; Zheng, Yuangang; Zhang, Yong-Wei; Han, Ming-Yong

    2015-05-20

    Here, we report a general and facile method for effective layer-by-layer exfoliation of transition metal dichalcogenides (TMDs) and graphite in water by using protein, bovine serum albumin (BSA) to produce single-layer nanosheets, which cannot be achieved using other commonly used bio- and synthetic polymers. Besides serving as an effective exfoliating agent, BSA can also function as a strong stabilizing agent against reaggregation of single-layer nanosheets for greatly improving their biocompatibility in biomedical applications. With significantly increased surface area, single-layer MoS2 nanosheets also exhibit a much higher binding capacity to pesticides and a much larger specific capacitance. The protein exfoliation process is carefully investigated with various control experiments and density functional theory simulations. It is interesting to find that the nonpolar groups of protein can firmly bind to TMD layers or graphene to expose polar groups in water, facilitating the effective exfoliation of single-layer nanosheets in aqueous solution. The present work will enable to optimize the fabrication of various 2D materials at high yield and large scale, and bring more opportunities to investigate the unique properties of 2D materials and exploit their novel applications. PMID:25936424

  15. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition

    PubMed Central

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  16. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition.

    PubMed

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  17. Kinetics of phase transition in protein solutions on microscopic and mesoscopic length scales

    NASA Astrophysics Data System (ADS)

    Filobelo, Luis F.

    2005-11-01

    Phase transformations in solutions of macromolecules are fundamental for all living things, and of great importance in science and industry. For instance, insulin is biosynthesized in the beta cells of the pancreas and stored in crystalline form, which protects it form cleavage, until it is needed. Certain diseases such as Alzheimer, sickle cell anemia, and eye cataract are produced by the polymerization of protein molecules, which loose their functionality after the phase transition. Additionally, separation operations in manufacturing of pharmaceuticals can be eliminated if the crystals produced have a narrow size distribution. The nucleation and growth of crystals can be adequately controlled only if the mechanisms that govern these processes are well understood. Here we have investigated several facets of the kinetics controlling the behavior of phase transition in protein solutions. We performed experiments to determine the homogenous nucleation rate for lysozyme and insulin crystals and the contribution of heterogeneously nucleated crystals. In the first segment of this work we discuss the existence of a solution-to-crystal spinodal boundary derived from these determinations, and showed that the formation of crystalline nuclei from solution occur in two steps for lysozyme: the formation of quasi-droplets of a disordered intermediate, followed by the nucleation of ordered crystalline embryos within these droplets in which the rate of each step depends on a respective free energy barrier and on the growth rate of its near-critical clusters. We addressed experimentally the relative significance of the free-energy barriers and the kinetic factors for the nucleation of crystals from solution. Using dynamic and static light scattering along with differential refractometry, we also characterized the appearance of dense liquid droplets and the magnitude of the second osmotic virial coefficient B2 for insulin in both aqueous solution and in solution containing 15% (v

  18. Protein-style dynamical transition in a non-biological polymer and a non-aqueous solvent

    DOE PAGESBeta

    Mamontov, E.; Sharma, V. K.; Borreguero, J. M.; Tyagi, M.

    2016-03-15

    Using neutron scattering and molecular dynamics simulation, techniques most often associated with protein dynamical transition studies, we have investigated the microscopic dynamics of one of the most common polymers, polystyrene, which was exposed to toluene vapor, mimicking the process of protein hydration from water vapor. Polystyrene with adsorbed toluene is an example of a solvent-solute system, which, unlike biopolymers, is anhydrous and lacks hydrogen bonding. Nevertheless, it exhibits the essential traits of the dynamical transition in biomolecules, such as a specific dependence of the microscopic dynamics of both solvent and host on the temperature and the amount of solvent adsorbed.more » Ultimately, we conclude that the protein dynamical transition is a manifestation of a universal solvent-solute dynamical relationship, which is not specific to either biomolecules as solute, or aqueous media as solvent, or even a particular type of interactions between solvent and solute.« less

  19. Hyperquenching and cold equilibration strategies for the study of liquid--liquid and protein folding transitions.

    PubMed

    Angell, C Austen; Wang, Li Min

    2003-09-01

    In this paper we consider the extension of the recent quantitative studies of hyperquenched glassformers to include (1). systems that exhibit first order liquid-liquid phase transitions, and (2). systems that contain molecules, which, during normal cooling, undergo internal structural changes above the glass temperature. The general aim of these studies is to trap-in a high enthalpy, high entropy, state of the system and then observe it evolving in time at low temperatures during a controlled annealing procedure. In this manner events that normally occur during change of temperature may be observed occurring during passage of time, at much lower temperatures. At such low temperatures the smearing effects of vibrations are greatly reduced. While the case of most interest in the second class is the refolding of thermally denatured protein molecules, any reconstructive molecular or chemical exchange process is a potential subject for investigation. Processes that occur in stages can be studied in greater detail, and any stage of interest can be frozen when desired, by drop of temperature, for more detailed spectroscopic examination. We review an electrospray method for hyperquenching liquids at approximately 10(5) K/s, and discuss some results of such experiments in order to illustrate a calorimetric approach to exploiting the hyperquenching-and- cold-equilibration strategy. To apply the idea to the study of proteins, the following protein solvent requirements must be met: (1). the solvents must not crystallize ice on cooling or heating, yet must not denature the proteins; (2). the solvents must support thermally denatured molecules without permitting aggregation. We describe two solvent systems, the first of which meets the first requirement, but the second only partially. The second solvent system apparently meets both. Preliminary results, only at the proof of concept stage, are reported for cold refolding of lysozyme, which, it seems, can be trapped in our solvent

  20. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition

    PubMed Central

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-01-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs. PMID:26358189

  1. The role of transit peptides in the localization of chloroplastic proteins: Progress report

    SciTech Connect

    Keegstra, K.

    1989-01-01

    Sjef Smeekens in the Utrecht group had just isolated a cDNA clone for PC and plans had been made for him to bring it to our lab to conduct import studies. In that application we proposed to examine the pathway of PC transport to the thylakoid lumen and to investigate the role of the transit peptide in that process. Additionally we proposed to begin studies aimed at investigating protein transport to the chloroplastic envelope. The progress made during the last three years in achieving these objectives is outlined below. During this grant period, we initiated some additional studies aimed at investigating the effect of known topogenic sequences on targeting within chloroplasts. Our results in these studies are also presented below. The descriptions provided in this report outline the major findings and conclusions.

  2. Efficient combination of Wang-Landau and transition matrix Monte Carlo methods for protein simulations.

    PubMed

    Ghulghazaryan, Ruben G; Hayryan, Shura; Hu, Chin-Kun

    2007-02-01

    An efficient combination of the Wang-Landau and transition matrix Monte Carlo methods for protein and peptide simulations is described. At the initial stage of simulation the algorithm behaves like the Wang-Landau algorithm, allowing to sample the entire interval of energies, and at the later stages, it behaves like transition matrix Monte Carlo method and has significantly lower statistical errors. This combination allows to achieve fast convergence to the correct values of density of states. We propose that the violation of TTT identities may serve as a qualitative criterion to check the convergence of density of states. The simulation process can be parallelized by cutting the entire interval of simulation into subintervals. The violation of ergodicity in this case is discussed. We test the algorithm on a set of peptides of different lengths and observe good statistical convergent properties for the density of states. We believe that the method is of general nature and can be used for simulations of other systems with either discrete or continuous energy spectrum. PMID:17195159

  3. Ordering Transitions Triggered by Specific Binding of Vesicles to Protein-Decorated Interfaces of Thermotropic Liquid Crystals

    PubMed Central

    Tan, Lie Na; Orler, Victor J.; Abbott, Nicholas L.

    2012-01-01

    We report that specific binding of ligand-functionalized (biotinylated) phospholipid vesicles (diameter = 120 ± 19 nm) to a monolayer of proteins (streptavidin or anti-biotin antibody) adsorbed at an interface between an aqueous phase and an immiscible film of a thermotropic liquid crystal (LC) (nematic 4′-pentyl-4-cyanobiphenyl (5CB)) triggers a continuous orientational ordering transition (continuous change in the tilt) in the LC. Results presented in this paper indicate that, following the capture of the vesicles at the LC interface via the specific binding interaction, phospholipids are transferred from the vesicles onto the LC interface to form a monolayer, reorganizing and partially displacing proteins from the LC interface. The dynamics of this process are accelerated substantially by the specific binding event relative to a protein-decorated interface of a LC that does not bind the ligands presented by vesicles. The observation of the continuous change in the ordering of the LC, when combined with other results presented in this paper, is significant as it is consistent with the presence of sub-optical domains of proteins and phospholipids on the LC interface. An additional significant hypothesis that emerges from the work reported in this paper is that the ordering transition of the LC is strongly influenced by the bound state of the protein adsorbed on the LC interface, as evidenced by the influence on the LC of (i) “crowding” of the protein within a monolayer formed at the LC interface and (ii) aging of the proteins on the LC interface. Overall, these results demonstrate that ordering transitions in LCs can be used to provide fundamental insights into the competitive adsorption of proteins and lipids at oil-water interfaces, and that LC ordering transitions have the potential to be useful for reporting specific binding events involving vesicles and proteins. PMID:22372743

  4. Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

    PubMed Central

    Baxa, Michael C.; Yu, Wookyung; Adhikari, Aashish N.; Ge, Liang; Xia, Zhen; Zhou, Ruhong; Freed, Karl F.; Sosnick, Tobin R.

    2015-01-01

    Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative β turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517–520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins. PMID:26100906

  5. Backbone and side chain chemical shift assignments of apolipophorin III from Galleria mellonella.

    PubMed

    Crowhurst, Karin A; Horn, James V C; Weers, Paul M M

    2016-04-01

    Apolipophorin III, a 163 residue monomeric protein from the greater wax moth Galleria mellonella (abbreviated as apoLp-IIIGM), has roles in upregulating expression of antimicrobial proteins as well as binding and deforming bacterial membranes. Due to its similarity to vertebrate apolipoproteins there is interest in performing atomic resolution analysis of apoLp-IIIGM as part of an effort to better understand its mechanism of action in innate immunity. In the first step towards structural characterization of apoLp-IIIGM, 99 % of backbone and 88 % of side chain (1)H, (13)C and (15)N chemical shifts were assigned. TALOS+ analysis of the backbone resonances has predicted that the protein is composed of five long helices, which is consistent with the reported structures of apolipophorins from other insect species. The next stage in the characterization of apoLp-III from G. mellonella will be to utilize these resonance assignments in solving the solution structure of this protein. PMID:26493308

  6. Protein-Style Dynamical Transition in a Non-Biological Polymer and a Non-Aqueous Solvent.

    PubMed

    Mamontov, E; Sharma, V K; Borreguero, J M; Tyagi, M

    2016-03-31

    Temperature-dependent onset of apparent anharmonicity in the microscopic dynamics of hydrated proteins and other biomolecules has been known as protein dynamical transition for the last quarter of a century. Using neutron scattering and molecular dynamics simulation, techniques most often associated with protein dynamical transition studies, we have investigated the microscopic dynamics of one of the most common polymers, polystyrene, which was exposed to toluene vapor, mimicking the process of protein hydration from water vapor. Polystyrene with adsorbed toluene is an example of a solvent-solute system, which, unlike biopolymers, is anhydrous and lacks hydrogen bonding. Nevertheless, it exhibits the essential traits of the dynamical transition in biomolecules, such as a specific dependence of the microscopic dynamics of both solvent and host on the temperature and the amount of solvent adsorbed. We conclude that the protein dynamical transition is a manifestation of a universal solvent-solute dynamical relationship, which is not specific to either biomolecules as solute, or aqueous media as solvent, or even a particular type of interactions between solvent and solute. PMID:26977709

  7. Triazole linkages and backbone branches in nucleic acids for biological and extra-biological applications

    NASA Astrophysics Data System (ADS)

    Paredes, Eduardo

    The recently increasing evidence of nucleic acids' alternative roles in biology and potential as useful nanomaterials and therapeutic agents has enabled the development of useful probes, elaborate nanostructures and therapeutic effectors based on nucleic acids. The study of alternative nucleic acid structure and function, particularly RNA, hinges on the ability to introduce site-specific modifications that either provide clues to the nucleic acid structure function relationship or alter the nucleic acid's function. Although the available chemistries allow for the conjugation of useful labels and molecules, their limitations lie in their tedious conjugation conditions or the lability of the installed probes. The development and optimization of click chemistry with RNA now provides the access to a robust and orthogonal conjugation methodology while providing stable conjugates. Our ability to introduce click reactive groups enzymatically, rather than only in the solid-phase, allows for the modification of larger, more cell relevant RNAs. Additionally, ligation of modified RNAs with larger RNA constructs through click chemistry represents an improvement over traditional ligation techniques. We determined that the triazole linkage generated through click chemistry is compatible in diverse nucleic acid based biological systems. Click chemistry has also been developed for extra-biological applications, particularly with DNA. We have expanded its use to generate useful polymer-DNA conjugates which can form controllable soft nanoparticles which take advantage of DNA's properties, i.e. DNA hybridization and computing. Additionally, we have generated protein-DNA conjugates and assembled protein-polymer hybrids mediated by DNA hybridization. The use of click chemistry in these reactions allows for the facile synthesis of these unnatural conjugates. We have also developed backbone branched DNA through click chemistry and showed that these branched DNAs are useful in generating

  8. The Graphical Representation of the Digital Astronaut Physiology Backbone

    NASA Technical Reports Server (NTRS)

    Briers, Demarcus

    2010-01-01

    This report summarizes my internship project with the NASA Digital Astronaut Project to analyze the Digital Astronaut (DA) physiology backbone model. The Digital Astronaut Project (DAP) applies integrated physiology models to support space biomedical operations, and to assist NASA researchers in closing knowledge gaps related to human physiologic responses to space flight. The DA physiology backbone is a set of integrated physiological equations and functions that model the interacting systems of the human body. The current release of the model is HumMod (Human Model) version 1.5 and was developed over forty years at the University of Mississippi Medical Center (UMMC). The physiology equations and functions are scripted in an XML schema specifically designed for physiology modeling by Dr. Thomas G. Coleman at UMMC. Currently it is difficult to examine the physiology backbone without being knowledgeable of the XML schema. While investigating and documenting the tags and algorithms used in the XML schema, I proposed a standard methodology for a graphical representation. This standard methodology may be used to transcribe graphical representations from the DA physiology backbone. In turn, the graphical representations can allow examination of the physiological functions and equations without the need to be familiar with the computer programming languages or markup languages used by DA modeling software.

  9. Determination of backbone chain direction of PDA using FFM

    NASA Astrophysics Data System (ADS)

    Jo, Sadaharu; Okamoto, Kentaro; Takenaga, Mitsuru

    2010-01-01

    The effect of backbone chains on friction force was investigated on both Langmuir-Blodgett (LB) films of 10,12-heptacosadiynoic acid and the (0 1 0) surfaces of single crystals of 2,4-hexadiene-1,6-diol using friction force microscopy (FFM). It was observed that friction force decreased when the scanning direction was parallel to the [0 0 1] direction in both samples. Moreover, friction force decreased when the scanning direction was parallel to the crystallographic [1 0 2], [1 0 1], [1 0 0] and [1 0 1¯] directions in only the single crystals. For the LB films, the [0 0 1] direction corresponds to the backbone chain direction of 10,12-heptacosadiynoic acid. For the single crystals, both the [0 0 1] and [1 0 1] directions correspond to the backbone chain direction, and the [1 0 2], [1 0 0] and [1 0 1¯] directions correspond to the low-index crystallographic direction. In both the LB films and single crystals, the friction force was minimized when the directions of scanning and the backbone chain were parallel.

  10. Impact of template backbone heterogeneity on RNA polymerase II transcription

    PubMed Central

    Xu, Liang; Wang, Wei; Zhang, Lu; Chong, Jenny; Huang, Xuhui; Wang, Dong

    2015-01-01

    Variations in the sugar component (ribose or deoxyribose) and the nature of the phosphodiester linkage (3′-5′ or 2′-5′ orientation) have been a challenge for genetic information transfer from the very beginning of evolution. RNA polymerase II (pol II) governs the transcription of DNA into precursor mRNA in all eukaryotic cells. How pol II recognizes DNA template backbone (phosphodiester linkage and sugar) and whether it tolerates the backbone heterogeneity remain elusive. Such knowledge is not only important for elucidating the chemical basis of transcriptional fidelity but also provides new insights into molecular evolution. In this study, we systematically and quantitatively investigated pol II transcriptional behaviors through different template backbone variants. We revealed that pol II can well tolerate and bypass sugar heterogeneity sites at the template but stalls at phosphodiester linkage heterogeneity sites. The distinct impacts of these two backbone components on pol II transcription reveal the molecular basis of template recognition during pol II transcription and provide the evolutionary insight from the RNA world to the contemporary ‘imperfect’ DNA world. In addition, our results also reveal the transcriptional consequences from ribose-containing genomic DNA. PMID:25662224

  11. Cooperative UAV-Based Communications Backbone for Sensor Networks

    SciTech Connect

    Roberts, R S

    2001-10-07

    The objective of this project is to investigate the use of unmanned air vehicles (UAVs) as mobile, adaptive communications backbones for ground-based sensor networks. In this type of network, the UAVs provide communication connectivity to sensors that cannot communicate with each other because of terrain, distance, or other geographical constraints. In these situations, UAVs provide a vertical communication path for the sensors, thereby mitigating geographic obstacles often imposed on networks. With the proper use of UAVs, connectivity to a widely disbursed sensor network in rugged terrain is readily achieved. Our investigation has focused on networks where multiple cooperating UAVs are used to form a network backbone. The advantage of using multiple UAVs to form the network backbone is parallelization of sensor connectivity. Many widely spaced or isolated sensors can be connected to the network at once using this approach. In these networks, the UAVs logically partition the sensor network into sub-networks (subnets), with one UAV assigned per subnet. Partitioning the network into subnets allows the UAVs to service sensors in parallel thereby decreasing the sensor-to-network connectivity. A UAV services sensors in its subnet by flying a route (path) through the subnet, uplinking data collected by the sensors, and forwarding the data to a ground station. An additional advantage of using multiple UAVs in the network is that they provide redundancy in the communications backbone, so that the failure of a single UAV does not necessarily imply the loss of the network.

  12. Tuning the Attempt Frequency of Protein Folding Dynamics via Transition-State Rigidification: Application to Trp-cage

    PubMed Central

    Abaskharon, Rachel M.; Culik, Robert M.; Woolley, G. Andrew; Gai, Feng

    2015-01-01

    The attempt frequency or prefactor (k0) of the transition state rate equation of protein folding kinetics has been estimated to be on the order of 106 s−1, which is many orders of magnitude smaller than that of chemical reactions. Herein, we use the mini-protein Trp-cage to show that it is possible to significantly increase the value of k0 for a protein folding reaction by rigidifying the transition state. This is achieved by reducing the conformational flexibility of a key structural element (i.e., an α-helix) formed in the transition state via photoisomerization of an azobenzene cross-linker. We find that this strategy not only decreases the folding time of the Trp-cage peptide by more than an order of magnitude (to ~100 ns at 25 °C) but also exposes parallel folding pathways, allowing us to provide, to the best of our knowledge, the first quantitative assessment of the curvature of the transition state free energy surface of a protein. PMID:26120378

  13. Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation.

    PubMed

    Chang, B S; Beauvais, R M; Dong, A; Carpenter, J F

    1996-07-15

    The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56 degrees C, during several weeks of storage at temperatures above and below the samples' Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 +/- 2.5 degrees C. With infrared spectroscopy, we determined that the protein lyophilized with /=5% sucrose, conformational change was inhibited during lyophilization. When stored at 50 degrees C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation. PMID:8660705

  14. Potential transition state analogue inhibitors for the penicillin-binding proteins.

    PubMed

    Pechenov, Aleksandr; Stefanova, Miglena E; Nicholas, Robert A; Peddi, Sridhar; Gutheil, William G

    2003-01-21

    Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis. The development of new PBP inhibitors is a potentially viable strategy for developing new antibacterial agents. Several potential transition state analogue inhibitors for the PBPs were synthesized, including peptide chloromethyl ketones, trifluoromethyl ketones, aldehydes, and boronic acids. These agents were characterized chemically, stereochemically, and as inhibitors of a set of low molecular mass PBPs: Escherichia coli (EC) PBP 5, Neisseria gonorrhoeae (NG) PBP 3, and NG PBP 4. A peptide boronic acid was the most effective PBP inhibitor in the series, with a preference observed for a d-boroAla-based over an l-boroAla-based inhibitor, as expected given that physiological PBP substrates are based on d-Ala at the cleavage site. The lowest K(I) of 370 nM was obtained for NG PBP 3 inhibition by Boc-l-Lys(Cbz)-d-boroAla (10b). Competitive inhibition was observed for this enzyme-inhibitor pair, as expected for an active site-directed inhibitor. For the three PBPs included in this study, an inverse correlation was observed between the values for log K(I) with 10b and the values for log(k(cat)/K(m)) for activity against the analogous substrate, and K(m)/K(I) ratios were 90, 1900, and 9600 for NG PBP 4, EC PBP 5, and NG PBP 3, respectively. These results demonstrate that peptide boronic acids can be effective transition state analogue inhibitors for the PBPs and provide a basis for the use of these agents as probes of PBP structure, function, and mechanism, as well as a possible basis for the development of new PBP-targeted antibacterial agents. PMID:12525187

  15. Backbone and side-chain (1)H, (15)N, (13)C assignment and secondary structure of BPSL1445 from Burkholderia pseudomallei.

    PubMed

    Quilici, Giacomo; Berardi, Andrea; Gaudesi, Davide; Gourlay, Louise J; Bolognesi, Martino; Musco, Giovanna

    2015-10-01

    BPSL1445 is a lipoprotein produced by the Gram-negative bacterium Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis. Immunodetection assays against sera patients using protein microarray suggest BPSL1445 involvement in melioidosis. Herein we report backbone, side chain NMR assignment and secondary structure for the recombinant protein. PMID:25893672

  16. Inorganic backbone ionomers: Design and dielectric response of single-ion conducting polymers

    NASA Astrophysics Data System (ADS)

    Bartels, Joshua

    analysis of the static dielectric constant and show excellent agreement with x-ray scattering and DFT calculations, each ionomer strongly favoring the formation of quadrupoles. Finally a polysiloxane ionomer was considered and was mixed with three anion and/or cation solvating additives, tetraglyme, tetraethylene glycol, and branched poly(ethylenimine). The EP model of the dielectric response gives the conducting ion concentration and the mobility of conducting ions and shows an increase in conducting ion concentration with both anion solvating and cation solvating additives. The static dielectric constant indicates an increased preference for ion pairs when anion receptors are present. Most interestingly, the additive that best decreased the glass transition temperature and increased the static dielectric constant did not result in the best dc conductivity. The best dc conductivity resulted from tetraglyme because it solvated cations without interacting with the polymer. High ion conductivities have not been observed in polymer systems that decouple charge transport from polymer motion, and therefore low Tg ionomers are the natural path forward for commercially viable ionomers. Inorganic backbone polymers impart a low Tg without bringing any strong disadvantage to ionomers. These materials are very important for developing superior ion conductors and should be pursued in future ionomer research.

  17. Inhibition of mitochondrial permeability transition by low pH is associated with less extensive membrane protein thiol oxidation.

    PubMed

    Teixeira, B M; Kowaltowski, A J; Castilho, R F; Vercesi, A E

    1999-12-01

    Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues. PMID:10841269

  18. Protein unfolding at interfaces: slow dynamics of alpha-helix to beta-sheet transition.

    PubMed

    Sethuraman, Ananthakrishnan; Vedantham, Ganesh; Imoto, Taiji; Przybycien, Todd; Belfort, Georges

    2004-09-01

    A two-phase sequential dynamic change in the secondary structure of hen egg lysozyme (Lys) adsorbed on solid substrates was observed. The first phase involved fast conversion of alpha-helix to random/turns (within the first minute or at very low coverage or high substrate wettability) with no perceptible change in beta-sheet content. The second phase (1-1200 min), however, involved a relatively slow conversion from alpha-helix to beta-sheet without a noticeable change in random/turns. An important finding of this work is that the concentration of lysozyme in the adsorbed state has a substantial effect on the fractional content of secondary structures. Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy, along with a newly-developed optimization algorithm for predicting the content of secondary structure motifs, was used to correlate the secondary structure and the amount of adsorbed lysozyme with the surface wettability of six different flat nanoporous substrates. Although three independent variables, surface wettability, solution concentration and time for adsorption, were used to follow the fractional structural changes of lysozyme, the results were all normalized onto a single plot with the amount adsorbed as the universal independent variable. Consequently, lateral interactions among proteins likely drive the transition process. Direct intermolecular force adhesion measurements between lysozyme and different functionalized self-assembled alkanethiol monolayers confirm that hydrophobic surfaces interact strongly with proteins. The lysozyme-unfolding pathway during early adsorption appears to be similar to that predicted by published molecular modeling results. PMID:15281120

  19. Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

    SciTech Connect

    Yu, Yuqi; Wang, Jinan; Shao, Qiang E-mail: Jiye.Shi@ucb.com Zhu, Weiliang E-mail: Jiye.Shi@ucb.com; Shi, Jiye E-mail: Jiye.Shi@ucb.com

    2015-03-28

    The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much less computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.

  20. Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

    NASA Astrophysics Data System (ADS)

    Yu, Yuqi; Wang, Jinan; Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2015-03-01

    The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much less computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.

  1. Structural Proteins from Whelk Egg Capsule with Long Range Elasticity Associated with a Solid-state Phase Transition

    PubMed Central

    Wasko, S. Scott; Tay, Gavin; Schwaighofer, Andreas; Nowak, Christoph; Waite, J. Herbert; Miserez, Ali

    2014-01-01

    The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helix to extended β-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle x-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules. PMID:24350603

  2. Effect of Liquid-Crystalline Epoxy Backbone Structure on Thermal Conductivity of Epoxy-Alumina Composites

    NASA Astrophysics Data System (ADS)

    Giang, Thanhkieu; Kim, Jinhwan

    2016-06-01

    In a series of papers published recently, we clearly demonstrated that the most important factor governing the thermal conductivity of epoxy-Al2O3 composites is the backbone structure of the epoxy. In this study, three more epoxies based on diglycidyl ester-terminated liquid-crystalline epoxy (LCE) have been synthesized to draw conclusions regarding the effect of the epoxy backbone structure on the thermal conductivity of epoxy-alumina composites. The synthesized structures were characterized by proton nuclear magnetic resonance (1H-NMR) and Fourier-transform infrared (FT-IR) spectroscopy. Differential scanning calorimetry, thermogravimetric analysis, and optical microscopy were also employed to examine the thermal and optical properties of the synthesized LCEs and the cured composites. All three LCE resins exhibited typical liquid-crystalline behaviors: clear solid crystalline state below the melting temperature (T m), sharp crystalline melting at T m, and transition to nematic phase above T m with consequent isotropic phase above the isotropic temperature (T i). The LCE resins displayed distinct nematic liquid-crystalline phase over a wide temperature range and retained liquid-crystalline phase after curing, with high thermal conductivity of the resulting composite. The thermal conductivity values ranged from 3.09 W/m-K to 3.89 W/m-K for LCE-Al2O3 composites with 50 vol.% filler loading. The steric effect played a governing role in the difference. The neat epoxy resin thermal conductivity was obtained as 0.35 W/m-K to 0.49 W/m-K based on analysis using the Agari-Uno model. The results clearly support the objective of this study in that the thermal conductivity of the LCE-containing networks strongly depended on the epoxy backbone structure and the degree of ordering in the cured network.

  3. Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control.

    PubMed

    Toledo-Núñez, Citlali; Vera-Robles, L Iraís; Arroyo-Maya, Izlia J; Hernández-Arana, Andrés

    2016-09-15

    A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models. PMID:27402175

  4. Molecular Dynamics Simulation on the Conformational Transition of the Mad2 Protein from the Open to the Closed State

    PubMed Central

    Li, Chaoqun; Zhu, Yanyan; Wang, Yan; Chen, Guangju

    2014-01-01

    The Mad2 protein, with two distinct conformations of open- and closed-states, is a key player in the spindle checkpoint. The closed Mad2 state is more active than the open one. We carried out conventional and targeted molecular dynamics simulations for the two stable Mad2 states and their conformational transition to address the dynamical transition mechanism from the open to the closed state. The intermediate structure in the transition process shows exposure of the β6 strand and an increase of space around the binding sites of β6 strand due to the unfolding of the β7/8 sheet and movement of the β6/4/5 sheet close to the αC helix. Therefore, Mad2 binding to the Cdc20 protein in the spindle checkpoint is made possible. The interconversion between these two states might facilitate the functional activity of the Mad2 protein. Motion correlation analysis revealed the allosteric network between the β1 strand and β7/8 sheet via communication of the β5-αC loop and the β6/4/5 sheet in this transition process. PMID:24690997

  5. Backbone Model of an Aquareovirus Virion by Cryo-Electron Microscopy and Bioinformatics

    PubMed Central

    Cheng, Lingpeng; Zhu, Jiang; Hui, Wong Hoi; Zhang, Xiaokang; Honig, Barry; Fang, Qin; Zhou, Z. Hong

    2010-01-01

    Grass carp reovirus (GCRV) is a member of the aquareovirus genus in the Reoviridae family and has a capsid with two shells—a transcription-competent core surrounded by a coat. We report a near-atomic-resolution reconstruction of the GCRV virion by cryo-electron microscopy and single-particle reconstruction. A backbone model of the GCRV virion, including seven conformers of the five capsid proteins making up the 1500 molecules in both the core and the coat, was derived using cryo-electron microscopy density-map-constrained homology modeling and refinement. Our structure clearly showed that the amino-terminal segment of core protein VP3B forms an ~120-Å-long α-helix-rich extension bridging across the icosahedral 2-fold-symmetry-related molecular interface. The presence of this unique structure across this interface and the lack of an external cementing molecule at this location in GCRV suggest a stabilizing role of this extended amino-terminal density. Moreover, part of this amino-terminal extension becomes invisible in the reconstruction of transcription-competent core particles, suggesting its involvement in endogenous viral RNA transcription. Our structure of the VP1 turret represents its open state, and comparison with its related structures at the closed state suggests hinge-like domain movements associated with the mRNA-capping machinery. Overall, this first backbone model of an aquareovirus virion provides a wealth of structural information for understanding the structural basis of GCRV assembly and transcription. PMID:20036256

  6. Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis.

    PubMed

    Zhang, Liguo; Zhang, Xiaofei; Ju, Hanxun; Chen, Jingui; Wang, Shucai; Wang, Hemeng; Zhao, Yuanling; Chang, Ying

    2016-02-12

    Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1-BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis. The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3-OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) in vivo. Further, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. These results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase. PMID:26809096

  7. Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach.

    PubMed

    Qayyum, Shariq; Sharma, Divakar; Bisht, Deepa; Khan, Asad U

    2016-06-10

    Enterococcus faecalis is a member of human gut microflora causing nosocomial infection involving biofilm formation. Ethyl methyl sulfonate induced mutants were analysed using crystal violet assay, SEM and CLSM microscopy which confirmed AK-E12 as biofilm efficient and AK-F6 as biofilm deficient mutants. Growth curve pattern revealed AK-E12 was fast growing whereas, AK-F6 was found slow growing mutant. 2D-Electrophorosis and MALDI-TOF analysis revealed over and underexpression of many translation-elongation associated proteins in mutants compared to wild type. Protein translation elongation factor G, translation elongation factor Tu and ribosomal subunit interface proteins were underexpressed and UTP-glucose-1-phosphate uridylyl transferase and cell division protein divIVA were overexpressed in AK-E12 as compared to wild type. In AK-F6, except 10 kDa chaperonin which was over-expressed other selected proteins were found to be suppressed. RT-PCR confirmed proteomic data except for the translation elongation factor G which showed contradictory data of proteome expression in AK-E12. Protein-protein interaction networks were constructed using STRING 10.0 which demonstrated strong connection of translation-elongation proteins with other proteins. Hence, it concludes from the data that translation elongation factors are important in transition of planktonic cells to biofilm cells in Enterococcus faecalis. PMID:27144316

  8. Identification of protein N-termini in Cyanophora paradoxa cyanelles: transit peptide composition and sequence determinants for precursor maturation

    PubMed Central

    Köhler, Daniel; Dobritzsch, Dirk; Hoehenwarter, Wolfgang; Helm, Stefan; Steiner, Jürgen M.; Baginsky, Sacha

    2015-01-01

    Glaucophyta, rhodophyta, and chloroplastida represent the three main evolutionary lineages that diverged from a common ancestor after primary endosymbiosis. Comparative analyses between members of these three lineages are a rich source of information on ancestral plastid features. We analyzed the composition and the cleavage site of cyanelle transit peptides from the glaucophyte Cyanophora paradoxa by terminal amine labeling of substrates (TAILS), and compared their characteristics to those of representatives of the chloroplastida. Our data show that transit peptide architecture is similar between members of these two lineages. This entails a comparable modular structure, an overrepresentation of serine or alanine and similarities in the amino acid composition around the processing peptidase cleavage site. The most distinctive difference is the overrepresentation of phenylalanine in the N-terminal 1–10 amino acids of cyanelle transit peptides. A quantitative proteome analysis with periplasm-free cyanelles identified 42 out of 262 proteins without the N-terminal phenylalanine, suggesting that the requirement for phenylalanine in the N-terminal region is not absolute. Proteins in this set are on average of low abundance, suggesting that either alternative import pathways are operating specifically for low abundance proteins or that the gene model annotation is incorrect for proteins with fewer EST sequences. We discuss these two possibilities and provide examples for both interpretations. PMID:26257763

  9. Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

    PubMed Central

    Chou, Ming-Lun; Chu, Chiung-Chih; Chen, Lih-Jen; Akita, Mitsuru; Li, Hsou-min

    2006-01-01

    Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation. PMID:17158958

  10. Observation of a dewetting transition in the collapse of the melittin tetramer

    NASA Astrophysics Data System (ADS)

    Liu, Pu; Huang, Xuhui; Zhou, Ruhong; Berne, B. J.

    2005-09-01

    Marked hydration changes occur during the self-assembly of the melittin protein tetramer in water. Hydrophobicity induces a drying transition in the gap between simple sufficiently large (more than 1nm2) strongly hydrophobic surfaces as they approach each other, resulting in the subsequent collapse of the system, as well as a depletion of water next to single surfaces. Here we investigate whether the hydrophobic induced collapse of multidomain proteins or the formation of protein oligimers exhibits a similar drying transition. We performed computer simulations to study the collapse of the tetramer of melittin in water, and observed a marked water drying transition inside a nanoscale channel of the tetramer (with a channel size of up to two or three water-molecule diameters). This transition, although occurring on a microscopic length scale, is analogous to a first-order phase transition from liquid to vapour. We find that this drying is very sensitive to single mutations of the three isoleucines to less hydrophobic residues and that such mutations in the right locations can switch the channel from being dry to being wet. Thus, quite subtle changes in hydrophobic surface topology can profoundly influence the drying transition. We show that, even in the presence of the polar protein backbone, sufficiently hydrophobic protein surfaces can induce a liquid-vapour transition providing an enormous driving force towards further collapse. This behaviour was unexpected because of the absence of drying in the collapse of the multidomain protein 2,3-dihydroxybiphenyl dioxygenase (BphC).

  11. On the role of thermal backbone fluctuations in myoglobin ligand gate dynamics

    NASA Astrophysics Data System (ADS)

    Krokhotin, Andrey; Niemi, Antti J.; Peng, Xubiao

    2013-05-01

    We construct an energy function that describes the crystallographic structure of sperm whale myoglobin backbone. As a model in our construction, we use the Protein Data Bank entry 1ABS that has been measured at liquid helium temperature. Consequently, the thermal B-factor fluctuations are very small, which is an advantage in our construction. The energy function that we utilize resembles that of the discrete nonlinear Schrödinger equation. Likewise, ours supports topological solitons as local minimum energy configurations. We describe the 1ABS backbone in terms of topological solitons with a precision that deviates from 1ABS by an average root-mean-square distance, which is less than the experimentally observed Debye-Waller B-factor fluctuation distance. We then subject the topological multi-soliton solution to extensive numerical heating and cooling experiments, over a very wide range of temperatures. We concentrate in particular to temperatures above 300 K and below the Θ-point unfolding temperature, which is around 348 K. We confirm that the behavior of the topological multi-soliton is fully consistent with Anfinsen's thermodynamic principle, up to very high temperatures. We observe that the structure responds to an increase of temperature consistently in a very similar manner. This enables us to characterize the onset of thermally induced conformational changes in terms of three distinct backbone ligand gates. One of the gates is made of the helix F and the helix E. The two other gates are chosen similarly, when open they provide a direct access route for a ligand to reach the heme. We find that out of the three gates we investigate, the one which is formed by helices B and G is the most sensitive to thermally induced conformational changes. Our approach provides a novel perspective to the important problem of ligand entry and exit.

  12. Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit

    PubMed Central

    Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry

    2015-01-01

    Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems. PMID:26506617

  13. Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

    DOE PAGESBeta

    Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry; Shehu, Amarda

    2015-10-27

    Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechainmore » conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.« less

  14. Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

    SciTech Connect

    Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry; Shehu, Amarda

    2015-10-27

    Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.

  15. Characterization of the structure and melting of DNAs containing backbone nicks and gaps

    SciTech Connect

    Snowden-Ifft, E.A.; Wemmer, D.E. )

    1990-06-26

    A DNA molecule containing a gap (a missing phosphate) has been examined and compared to two other molecules of the same sequence, one containing a nick (a phosphorylated gap) and the other a normal duplex containing no break in the backbone. A second gapped sequence was also compared to a normal duplex of the same sequence. The molecules containing nicks or gaps were generated as dumbbell molecules, short helices closed by a loop at each end. The dumbbells were formed by the association of two hairpins with self-complementary dangling 5'-ends. Nuclear magnetic resonance was used to monitor the melting transition and to probe structural differences between molecules. Under the conditions used here no change in stability was observed upon phosphorylation of the gap. Structural changes upon phosphorylation of a gap or closure of a nick were minimal and were localized to the region immediately around the gap or nick. Two transitions can be observed as a gapped or nicked molecule melts, although the resolution of the two transitions varies with the salt concentration. At moderate to high salt (greater than or equal to 30 mM) the molecule melts essentially all at once. At low salt the two transitions occur at temperatures that differ by as much as 15 degrees C. In addition, comparison with other NMR melting studies indicates that the duplex formed by the overlap of the dangling ends of the hairpins is stabilized relative to a free duplex of the same sequence, probably by stacking onto the hairpin stem.

  16. A note on protein expression changes in chicken breast muscle in response to time in transit before slaughtering

    PubMed Central

    2013-01-01

    Aims of the research were to devise a proteome map of the chicken Pectoralis superficialis muscle, as resolved by two-dimensional gel electrophoresis, and to characterize protein expression changes in the soluble protein fraction in commercial conditions due to age and to time in transit before slaughtering. Broilers were reared under commercial conditions until they reached a mean 1.8 kg and 36 d, or 2.6 kg and 46 d of age. Transport to the slaughterhouse took 90 or 220 minutes. Transport-induced stress was assessed from blood metabolites and leukocyte cell counts, revealing significant changes in albumin, glucose and triglyceride concentrations, in heterophils and leukocyte counts for chickens in transit for longer, and in glucose depending mainly on age. The sarcoplasmic protein fractions were extracted from a total of 39 breast muscle samples, collected 15 min post mortem, for analysis by two-dimensional electrophoresis. Image and statistical analyses enabled us to study the qualitative and quantitative differences between the samples. Twelve up- or down-regulated protein spots were detected (P < 0.05): 8 related to the age effect, 2 to time in transit, and 2 to the interaction between the two. Age and time in transit influenced the avian proteome regulating the biological processes linked to the cellular housekeeping functions, related mainly to metabolism, cell division and control of apoptosis. Principal component analysis clustering was used to assess differences between birds. Age difference discriminated between the chickens analyzed better than time in transit, which seemed to have less general impact on the proteome fraction considered here. Isolating and identifying the proteins whose expression changes in response to transport duration and age shed some light on the biological mechanisms underlying growth and stress-related metabolism in chickens. Our results, combined with a further characterization of the chicken proteome associated with

  17. Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors.

    PubMed

    Kunduri, Govind; Yuan, Changqing; Parthibane, Velayoudame; Nyswaner, Katherine M; Kanwar, Ritu; Nagashima, Kunio; Britt, Steven G; Mehta, Nickita; Kotu, Varshika; Porterfield, Mindy; Tiemeyer, Michael; Dolph, Patrick J; Acharya, Usha; Acharya, Jairaj K

    2014-07-01

    The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking. PMID:25002678

  18. Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

    PubMed Central

    Spraggon, Glen; Koesema, Eric; Scarselli, Maria; Malito, Enrico; Biagini, Massimiliano; Norais, Nathalie; Emolo, Carla; Barocchi, Michèle Anne; Giusti, Fabiola; Hilleringmann, Markus; Rappuoli, Rino; Lesley, Scott; Covacci, Antonello; Masignani, Vega; Ferlenghi, Ilaria

    2010-01-01

    Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility. PMID:20559564

  19. RNA-Redesign: a web server for fixed-backbone 3D design of RNA.

    PubMed

    Yesselman, Joseph D; Das, Rhiju

    2015-07-01

    RNA is rising in importance as a design medium for interrogating fundamental biology and for developing therapeutic and bioengineering applications. While there are several online servers for design of RNA secondary structure, there are no tools available for the rational design of 3D RNA structure. Here we present RNA-Redesign (http://rnaredesign.stanford.edu), an online 3D design tool for RNA. This resource utilizes fixed-backbone design to optimize the sequence identity and nucleobase conformations of an RNA to match a desired backbone, analogous to fundamental tools that underlie rational protein engineering. The resulting sequences suggest thermostabilizing mutations that can be experimentally verified. Further, sequence preferences that differ between natural and computationally designed sequences can suggest whether natural sequences possess functional constraints besides folding stability, such as cofactor binding or conformational switching. Finally, for biochemical studies, the designed sequences can suggest experimental tests of 3D models, including concomitant mutation of base triples. In addition to the designs generated, detailed graphical analysis is presented through an integrated and user-friendly environment. PMID:25964298

  20. RNA-Redesign: a web server for fixed-backbone 3D design of RNA

    PubMed Central

    Yesselman, Joseph D.; Das, Rhiju

    2015-01-01

    RNA is rising in importance as a design medium for interrogating fundamental biology and for developing therapeutic and bioengineering applications. While there are several online servers for design of RNA secondary structure, there are no tools available for the rational design of 3D RNA structure. Here we present RNA-Redesign (http://rnaredesign.stanford.edu), an online 3D design tool for RNA. This resource utilizes fixed-backbone design to optimize the sequence identity and nucleobase conformations of an RNA to match a desired backbone, analogous to fundamental tools that underlie rational protein engineering. The resulting sequences suggest thermostabilizing mutations that can be experimentally verified. Further, sequence preferences that differ between natural and computationally designed sequences can suggest whether natural sequences possess functional constraints besides folding stability, such as cofactor binding or conformational switching. Finally, for biochemical studies, the designed sequences can suggest experimental tests of 3D models, including concomitant mutation of base triples. In addition to the designs generated, detailed graphical analysis is presented through an integrated and user-friendly environment. PMID:25964298

  1. Morphine-Induced Preconditioning: Involvement of Protein Kinase A and Mitochondrial Permeability Transition Pore

    PubMed Central

    Dorsch, Marianne; Behmenburg, Friederike; Raible, Miriam; Blase, Dominic; Grievink, Hilbert; Hollmann, Markus W.; Heinen, André; Huhn, Ragnar

    2016-01-01

    Background Morphine induces myocardial preconditioning (M-PC) via activation of mitochondrial large conductance Ca2+-sensitive potassium (mKCa) channels. An upstream regulator of mKCa channels is protein kinase A (PKA). Furthermore, mKCa channel activation regulates mitochondrial bioenergetics and thereby prevents opening of the mitochondrial permeability transition pore (mPTP). Here, we investigated in the rat heart in vivo whether 1) M-PC is mediated by activation of PKA, and 2) pharmacological opening of the mPTP abolishes the cardioprotective effect of M-PC and 3) M-PC is critically dependent on STAT3 activation, which is located upstream of mPTP within the signalling pathway. Methods Male Wistar rats were randomised to six groups (each n = 6). All animals underwent 25 minutes of regional myocardial ischemia and 120 minutes of reperfusion. Control animals (Con) were not further treated. Morphine preconditioning was initiated by intravenous administration of 0.3 mg/kg morphine (M-PC). The PKA blocker H-89 (10 μg/kg) was investigated with and without morphine (H-89+M-PC, H-89). We determined the effect of mPTP opening with atractyloside (5 mg/kg) with and without morphine (Atr+M-PC, Atr). Furthermore, the effect of morphine on PKA activity was tested in isolated adult rat cardiomyocytes. In further experiments in isolated hearts we tested the protective properties of morphine in the presence of STAT3 inhibition, and whether pharmacological prevention of the mPTP-opening by cyclosporine A (CsA) is cardioprotective in the presence of STAT3 inhibition. Results Morphine reduced infarct size from 64±5% to 39±9% (P<0.05 vs. Con). H-89 completely blocked preconditioning by morphine (64±9%; P<0.05 vs. M-PC), but H-89 itself had not effect on infarct size (61±10%; P>0.05 vs. Con). Also, atractyloside abolished infarct size reduction of morphine completely (65±9%; P<0.05 vs. M-PC) but had no influence on infarct size itself (64±5%; P>0.05 vs. Con). In isolated

  2. Elucidating the ensemble of functionally-relevant transitions in protein systems with a robotics-inspired method

    PubMed Central

    2013-01-01

    Background Many proteins tune their biological function by transitioning between different functional states, effectively acting as dynamic molecular machines. Detailed structural characterization of transition trajectories is central to understanding the relationship between protein dynamics and function. Computational approaches that build on the Molecular Dynamics framework are in principle able to model transition trajectories at great detail but also at considerable computational cost. Methods that delay consideration of dynamics and focus instead on elucidating energetically-credible conformational paths connecting two functionally-relevant structures provide a complementary approach. Effective sampling-based path planning methods originating in robotics have been recently proposed to produce conformational paths. These methods largely model short peptides or address large proteins by simplifying conformational space. Methods We propose a robotics-inspired method that connects two given structures of a protein by sampling conformational paths. The method focuses on small- to medium-size proteins, efficiently modeling structural deformations through the use of the molecular fragment replacement technique. In particular, the method grows a tree in conformational space rooted at the start structure, steering the tree to a goal region defined around the goal structure. We investigate various bias schemes over a progress coordinate for balance between coverage of conformational space and progress towards the goal. A geometric projection layer promotes path diversity. A reactive temperature scheme allows sampling of rare paths that cross energy barriers. Results and conclusions Experiments are conducted on small- to medium-size proteins of length up to 214 amino acids and with multiple known functionally-relevant states, some of which are more than 13Å apart of each-other. Analysis reveals that the method effectively obtains conformational paths connecting

  3. Backbone Structure of Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli

    PubMed Central

    Hilger, Daniel; Polyhach, Yevhen; Jung, Heinrich; Jeschke, Gunnar

    2009-01-01

    Abstract The backbone structure is determined by site-directed spin labeling, double electron electron resonance measurements of distances, and modeling in terms of a helix-loop-helix construct for a transmembrane domain that is supposed to line the translocation pathway in the 54.3 kDa Na+/proline symporter PutP of Escherichia coli. The conformational distribution of the spin labels is accounted for by a rotamer library. An ensemble of backbone models with a root mean-square deviation of less than 2 Å is obtained. These models exhibit a pronounced kink near residue T341, which is involved in substrate binding. The kink may be associated with a hinge that allows the protein to open and close an inwardly oriented cavity. PMID:19134477

  4. Picosecond conformational transition and equilibration of a cyclic peptide

    NASA Astrophysics Data System (ADS)

    Bredenbeck, Jens; Helbing, Jan; Sieg, Arne; Schrader, Tobias; Zinth, Wolfgang; Renner, Christian; Behrendt, Raymond; Moroder, Luis; Wachtveitl, Josef; Hamm, Peter

    2003-05-01

    Ultrafast IR spectroscopy is used to monitor the nonequilibrium backbone dynamics of a cyclic peptide in the amide I vibrational range with picosecond time resolution. A conformational change is induced by means of a photoswitch integrated into the peptide backbone. Although the main conformational change of the backbone is completed after only 20 ps, the subsequent equilibration in the new region of conformational space continues for times >16 ns. Relaxation and equilibration processes of the peptide backbone occur on a discrete hierarchy of time scales. Albeit possessing only a few conformational degrees of freedom compared with a protein, the peptide behaves highly nontrivially and provides insights into the complexity of fast protein folding.

  5. Robust identification of backbone curves using control-based continuation

    NASA Astrophysics Data System (ADS)

    Renson, L.; Gonzalez-Buelga, A.; Barton, D. A. W.; Neild, S. A.

    2016-04-01

    Control-based continuation is a recently developed approach for testing nonlinear dynamic systems in a controlled manner and exploring their dynamic features as system parameters are varied. In this paper, control-based continuation is adapted to follow the locus where system response and excitation are in quadrature, extracting the backbone curve of the underlying conservative system. The method is applied to a single-degree-of-freedom oscillator under base excitation, and the results are compared with the standard resonant-decay method.

  6. Scientific Laser Market Still The Backbone Of The Industry

    NASA Astrophysics Data System (ADS)

    Tomkins, Jon

    1988-06-01

    Good morning. I would like to thank Gary Forrest and Laser Focus for giving me the opportunity to participate in this seminar. After listening to Moe Levitt's overview of the industry outlook for 1988, it is a pleasure for me to represent one of the 'hot' sectors in the market - the old research market. My mission today is to provide a perspective of this market to you as one of the healthier parts of the industry, as one of the backbones of our industry.

  7. Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

    PubMed

    Zaitsev, Boris N; Benedetti, Fabrizio; Mikhaylov, Andrey G; Korneev, Denis V; Sekatskii, Sergey K; Karakouz, Tanya; Belavin, Pavel A; Netesova, Nina A; Protopopova, Elena V; Konovalova, Svetlana N; Dietler, Giovanni; Loktev, Valery B

    2014-12-01

    The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. PMID:25319621

  8. Protein Brownian Rotation at the Glass Transition Temperature of a Freeze-Concentrated Buffer Probed by Superparamagnetic Nanoparticles

    PubMed Central

    Eloi, J.-C.; Okuda, M.; Jones, S.E. Ward; Schwarzacher, W.

    2013-01-01

    For applications from food science to the freeze-thawing of proteins it is important to understand the often complex freezing behavior of solutions of biomolecules. Here we use a magnetic method to monitor the Brownian rotation of a quasi-spherical cage-shaped protein, apoferritin, approaching the glass transition Tg in a freeze-concentrated buffer (Tris-HCl). The protein incorporates a synthetic magnetic nanoparticle (Co-doped Fe3O4 (magnetite)). We use the magnetic signal from the nanoparticles to monitor the protein orientation. As T decreases toward Tg of the buffer solution the protein’s rotational relaxation time increases exponentially, taking values in the range from a few seconds up to thousands of seconds, i.e., orders of magnitude greater than usually accessed, e.g., by NMR. The longest relaxation times measured correspond to estimated viscosities >2 MPa s. As well as being a means to study low-temperature, high-viscosity environments, our method provides evidence that, for the cooling protocol used, the following applies: 1), the concentration of the freeze-concentrated buffer at Tg is independent of its initial concentration; 2), little protein adsorption takes place at the interface between ice and buffer; and 3), the protein is free to rotate even at temperatures as low as 207 K. PMID:23790376

  9. Effect of hydrolyzed whey protein on surface morphology, water sorption, and glass transition temperature of a model infant formula.

    PubMed

    Kelly, Grace M; O'Mahony, James A; Kelly, Alan L; O'Callaghan, Donal J

    2016-09-01

    Physical properties of spray-dried dairy powders depend on their composition and physical characteristics. This study investigated the effect of hydrolyzed whey protein on the microstructure and physical stability of dried model infant formula. Model infant formulas were produced containing either intact (DH 0) or hydrolyzed (DH 12) whey protein, where DH=degree of hydrolysis (%). Before spray drying, apparent viscosities of liquid feeds (at 55°C) at a shear rate of 500 s(-1) were 3.02 and 3.85 mPa·s for intact and hydrolyzed infant formulas, respectively. On reconstitution, powders with hydrolyzed whey protein had a significantly higher fat globule size and lower emulsion stability than intact whey protein powder. Lactose crystallization in powders occurred at higher relative humidity for hydrolyzed formula. The Guggenheim-Anderson-de Boer equation, fitted to sorption isotherms, showed increased monolayer moisture when intact protein was present. As expected, glass transition decreased significantly with increasing water content. Partial hydrolysis of whey protein in model infant formula resulted in altered powder particle surface morphology, lactose crystallization properties, and storage stability. PMID:27320674

  10. CP29, a monomeric light-harvesting complex II protein, is essential for state transitions in Chlamydomonas reinhardtii.

    PubMed

    Tokutsu, Ryutaro; Iwai, Masakazu; Minagawa, Jun

    2009-03-20

    In oxygen-evolving photosynthesis, the two photosystems, photosystem I (PSI) and photosystem II (PSII), function in parallel, and their excitation levels must be balanced to maintain an optimal photosynthetic rate under various light conditions. State transitions balance excitation energy between the two photosystems by redistributing light-harvesting complex II (LHCII) proteins. Here we describe two RNA interference (RNAi) mutants of the green alga Chlamydomonas reinhardtii with one of the minor monomeric LHCII proteins, CP29 or CP26, knocked down. These two proteins have been identified in PSI-LHCI supercomplexes that harbor mobile LHCII proteins from PSII under a state where PSII is preferentially excited (State 2). We show that both the CP29 and CP26 RNAi mutants undergo reductions in the PSII antenna size during a transition from State 1 (a state where PSI is preferentially excited) to State 2, as reflected by nonphotochemical quenching of fluorescence, low temperature fluorescence spectra, and functional absorption cross-section. However, the undocked LHCIIs from PSII do not re-associate with PSI in the CP29-RNAi (b4i) mutant because the antenna size of PSI was not complementary increased. The mobile LHCIIs in the CP26-RNAi (b5i) mutant, however, re-associate with PSI, whose PSI-LHCI/II supercomplex is visualized on a sucrose density gradient. This study clarifies that CP29, not CP26, is an essential component in state transitions and demonstrates that CP29 is crucial when mobile LHCIIs re-associate with PSI under State 2 conditions. PMID:19144643

  11. Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis

    DOE PAGESBeta

    Zhang, Liguo; Zhang, Xiaofei; Ju, Hanxun; Chen, Jingui; Wang, Shucai; Wang, Hemeng; Zhao, Yuanling; Chang, Ying

    2016-01-23

    We study the Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 that regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1–BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis. The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3–OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) inmore » vivo. In addition, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. In conclusion, these results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase.« less

  12. Surface tension effects on the phase transition of a DPPC bilayer with and without protein: a molecular dynamics simulation.

    PubMed

    Kong, Xian; Qin, Shanshan; Lu, Diannan; Liu, Zheng

    2014-05-14

    While the surface tension of a cell membrane, or a plasma membrane, regulates cell functions, little is known about its effect on the conformational changes of the lipid bilayer and hence the resulting changes in the cell membrane. To obtain some insights into the phase transition of the lipid bilayer as a function of surface tension, we used a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer as a model lipid bilayer and aquaporin (AqpZ), a transmembrane channel protein for water, as a model embedded protein. A coarse-grained molecular dynamics simulation was applied to illustrate the phase transition behavior of the pure DPPC bilayer and aquaporin-embedded DPPC bilayer under different surface tensions. It was shown that an increased surface tension reduced the phase transition temperature of the DPPC bilayer. As for the DPPC bilayer in gel form, no significant changes occurred in the structure of the bilayer in response to the surface tension. Once in a liquid crystal state, both the structure and properties of the DPPC bilayer, such as area per lipid, lipid order parameters, bilayer thickness and lateral diffusion coefficients, were responsive to the magnitude of surface tension in a linear way. The presence of aquaporin attenuated the compact alignment of the lipid bilayer, hindered the parallel movement, and thus made the DPPC bilayer less sensitive to the surface tension. PMID:24668218

  13. Conformational Transitions in the Membrane Scaffold Protein of Phospholipid Bilayer Nanodiscs*

    PubMed Central

    Morgan, Christopher R.; Hebling, Christine M.; Rand, Kasper D.; Stafford, Darrel W.; Jorgenson, James W.; Engen, John R.

    2011-01-01

    Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest. PMID:21715319

  14. Solid State Nuclear Magnetic Resonance Investigation of Polymer Backbone Dynamics in Poly(Ethylene Oxide) Based Lithium and Sodium Polyether-ester-sulfonate Ionomers

    SciTech Connect

    Roach, David J.; Dou, Shichen; Colby, Ralph H.; Mueller, Karl T.

    2013-01-01

    Polymer backbone dynamics of single ion conducting poly(ethylene oxide) (PEO)-based ionomer samples with low glass transition temperatures (Tg) have been investigated using solid-state nuclear magnetic resonance (NMR). Experiments detecting 13C with 1H decoupling under magic angle spinning (MAS) conditions identified the different components of the polymer backbone (PEO spacer and isophthalate groups) and their relative mobilities for a suite of lithium- and sodium-containing ionomer samples with varying cation contents. Variable temperature (203-373 K) 1H-13C cross-polarization MAS (CP-MAS) experiments also provided qualitative assessment of the differences in the motions of the polymer backbone components as a function of cation content and identity. Each of the main backbone components exhibit distinct motions, following the trends expected for motional characteristics based on earlier Quasi Elastic Neutron Scattering and 1H spin-lattice relaxation rate measurements. Previous 1H and 7Li spin-lattice relaxation measurements focused on both the polymer backbone and cation motion on the nanosecond timescale. The studies presented here assess the slower timescale motion of the polymer backbone allowing for a more comprehensive understanding of the polymer dynamics. The temperature dependences of 13C linewidths were used to both qualitatively and quantitatively examine the effects of cation content and identity on PEO spacer mobility. Variable contact time 1H-13C CP-MAS experiments were used to further assess the motions of the polymer backbone on the microsecond timescale. The motion of the PEO spacer, reported via the rate of magnetization transfer from 1H to 13C nuclei, becomes similar for T ≳ 1.1 Tg in all ionic samples, indicating that at similar elevated reduced temperatures the motions of the polymer backbones on the microsecond timescale become insensitive to ion interactions. These results present an improved picture, beyond those of previous findings, for

  15. Solid state nuclear magnetic resonance investigation of polymer backbone dynamics in poly(ethylene oxide) based lithium and sodium polyether-ester-sulfonate ionomers

    NASA Astrophysics Data System (ADS)

    Roach, David J.; Dou, Shichen; Colby, Ralph H.; Mueller, Karl T.

    2013-05-01

    Polymer backbone dynamics of single ion conducting poly(ethylene oxide) (PEO)-based ionomer samples with low glass transition temperatures (Tg) have been investigated using solid-state nuclear magnetic resonance. Experiments detecting 13C with 1H decoupling under magic angle spinning (MAS) conditions identified the different components of the polymer backbone (PEO spacer and isophthalate groups) and their relative mobilities for a suite of lithium- and sodium-containing ionomer samples with varying cation contents. Variable temperature (203-373 K) 1H-13C cross-polarization MAS (CP-MAS) experiments also provided qualitative assessment of the differences in the motions of the polymer backbone components as a function of cation content and identity. Each of the main backbone components exhibit distinct motions, following the trends expected for motional characteristics based on earlier Quasi Elastic Neutron Scattering and 1H spin-lattice relaxation rate measurements. Previous 1H and 7Li spin-lattice relaxation measurements focused on both the polymer backbone and cation motion on the nanosecond timescale. The studies presented here assess the slower timescale motion of the polymer backbone allowing for a more comprehensive understanding of the polymer dynamics. The temperature dependences of 13C linewidths were used to both qualitatively and quantitatively examine the effects of cation content and identity on PEO spacer mobility. Variable contact time 1H-13C CP-MAS experiments were used to further assess the motions of the polymer backbone on the microsecond timescale. The motion of the PEO spacer, reported via the rate of magnetization transfer from 1H to 13C nuclei, becomes similar for T ˜x 1{.1} Tg in all ionic samples, indicating that at similar elevated reduced temperatures the motions of the polymer backbones on the microsecond timescale become insensitive to ion interactions. These results present an improved picture, beyond those of previous findings, for the

  16. Exploring percolative landscapes: Infinite cascades of geometric phase transitions

    NASA Astrophysics Data System (ADS)

    Timonin, P. N.; Chitov, Gennady Y.

    2016-01-01

    The evolution of many kinetic processes in 1+1 (space-time) dimensions results in 2 D directed percolative landscapes. The active phases of these models possess numerous hidden geometric orders characterized by various types of large-scale and/or coarse-grained percolative backbones that we define. For the patterns originated in the classical directed percolation (DP) and contact process we show from the Monte Carlo simulation data that these percolative backbones emerge at specific critical points as a result of continuous phase transitions. These geometric transitions belong to the DP universality class and their nonlocal order parameters are the capacities of corresponding backbones. The multitude of conceivable percolative backbones implies the existence of infinite cascades of such geometric transitions in the kinetic processes considered. We present simple arguments to support the conjecture that such cascades of transitions are a generic feature of percolation as well as of many other transitions with nonlocal order parameters.

  17. Exploring percolative landscapes: Infinite cascades of geometric phase transitions.

    PubMed

    Timonin, P N; Chitov, Gennady Y

    2016-01-01

    The evolution of many kinetic processes in 1+1 (space-time) dimensions results in 2D directed percolative landscapes. The active phases of these models possess numerous hidden geometric orders characterized by various types of large-scale and/or coarse-grained percolative backbones that we define. For the patterns originated in the classical directed percolation (DP) and contact process we show from the Monte Carlo simulation data that these percolative backbones emerge at specific critical points as a result of continuous phase transitions. These geometric transitions belong to the DP universality class and their nonlocal order parameters are the capacities of corresponding backbones. The multitude of conceivable percolative backbones implies the existence of infinite cascades of such geometric transitions in the kinetic processes considered. We present simple arguments to support the conjecture that such cascades of transitions are a generic feature of percolation as well as of many other transitions with nonlocal order parameters. PMID:26871019

  18. A phylogenetic backbone for Bivalvia: an RNA-seq approach

    PubMed Central

    González, Vanessa L.; Andrade, Sónia C. S.; Bieler, Rüdiger; Collins, Timothy M.; Dunn, Casey W.; Mikkelsen, Paula M.; Taylor, John D.; Giribet, Gonzalo

    2015-01-01

    Bivalves are an ancient and ubiquitous group of aquatic invertebrates with an estimated 10 000–20 000 living species. They are economically significant as a human food source, and ecologically important given their biomass and effects on communities. Their phylogenetic relationships have been studied for decades, and their unparalleled fossil record extends from the Cambrian to the Recent. Nevertheless, a robustly supported phylogeny of the deepest nodes, needed to fully exploit the bivalves as a model for testing macroevolutionary theories, is lacking. Here, we present the first phylogenomic approach for this important group of molluscs, including novel transcriptomic data for 31 bivalves obtained through an RNA-seq approach, and analyse these data with published genomes and transcriptomes of other bivalves plus outgroups. Our results provide a well-resolved, robust phylogenetic backbone for Bivalvia with all major lineages delineated, addressing long-standing questions about the monophyly of Protobranchia and Heterodonta, and resolving the position of particular groups such as Palaeoheterodonta, Archiheterodonta and Anomalodesmata. This now fully resolved backbone demonstrates that genomic approaches using hundreds of genes are feasible for resolving phylogenetic questions in bivalves and other animals. PMID:25589608

  19. Long-term forecasting of internet backbone traffic.

    PubMed

    Papagiannaki, Konstantina; Taft, Nina; Zhang, Zhi-Li; Diot, Christophe

    2005-09-01

    We introduce a methodology to predict when and where link additions/upgrades have to take place in an Internet protocol (IP) backbone network. Using simple network management protocol (SNMP) statistics, collected continuously since 1999, we compute aggregate demand between any two adjacent points of presence (PoPs) and look at its evolution at time scales larger than 1 h. We show that IP backbone traffic exhibits visible long term trends, strong periodicities, and variability at multiple time scales. Our methodology relies on the wavelet multiresolution analysis (MRA) and linear time series models. Using wavelet MRA, we smooth the collected measurements until we identify the overall long-term trend. The fluctuations around the obtained trend are further analyzed at multiple time scales. We show that the largest amount of variability in the original signal is due to its fluctuations at the 12-h time scale. We model inter-PoP aggregate demand as a multiple linear regression model, consisting of the two identified components. We show that this model accounts for 98% of the total energy in the original signal, while explaining 90% of its variance. Weekly approximations of those components can be accurately modeled with low-order autoregressive integrated moving average (ARIMA) models. We show that forecasting the long term trend and the fluctuations of the traffic at the 12-h time scale yields accurate estimates for at least 6 months in the future. PMID:16252820

  20. A phylogenetic backbone for Bivalvia: an RNA-seq approach.

    PubMed

    González, Vanessa L; Andrade, Sónia C S; Bieler, Rüdiger; Collins, Timothy M; Dunn, Casey W; Mikkelsen, Paula M; Taylor, John D; Giribet, Gonzalo

    2015-02-22

    Bivalves are an ancient and ubiquitous group of aquatic invertebrates with an estimated 10 000-20 000 living species. They are economically significant as a human food source, and ecologically important given their biomass and effects on communities. Their phylogenetic relationships have been studied for decades, and their unparalleled fossil record extends from the Cambrian to the Recent. Nevertheless, a robustly supported phylogeny of the deepest nodes, needed to fully exploit the bivalves as a model for testing macroevolutionary theories, is lacking. Here, we present the first phylogenomic approach for this important group of molluscs, including novel transcriptomic data for 31 bivalves obtained through an RNA-seq approach, and analyse these data with published genomes and transcriptomes of other bivalves plus outgroups. Our results provide a well-resolved, robust phylogenetic backbone for Bivalvia with all major lineages delineated, addressing long-standing questions about the monophyly of Protobranchia and Heterodonta, and resolving the position of particular groups such as Palaeoheterodonta, Archiheterodonta and Anomalodesmata. This now fully resolved backbone demonstrates that genomic approaches using hundreds of genes are feasible for resolving phylogenetic questions in bivalves and other animals. PMID:25589608

  1. Sequential backbone resonance assignments of the E. coli dihydrofolate reductase Gly67Val mutant: folate complex.

    PubMed

    Puthenpurackal Narayanan, Sunilkumar; Maeno, Akihiro; Wada, Yuji; Tate, Shin-Ichi; Akasaka, Kazuyuki

    2016-04-01

    Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(β) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35 °C, and then evaluated the H(N), N(H), C(α) and C(β) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain. PMID:26482924

  2. Side chain and backbone contributions of Phe508 to CFTR folding

    SciTech Connect

    Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J.

    2010-12-07

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

  3. Functional role of a protein foldon--an Omega-loop foldon controls the alkaline transition in ferricytochrome c.

    PubMed

    Maity, Haripada; Rumbley, Jon N; Englander, S Walter

    2006-05-01

    Hydrogen exchange results for cytochrome c and several other proteins show that they are composed of a number of foldon units which continually unfold and refold and account for some functional properties. Previous work showed that one Omega-loop foldon controls the rate of the structural switching and ligand exchange behavior of cytochrome c known as the alkaline transition. The present work tests the role of foldons in the alkaline transition equilibrium. We measured the effects of denaturant and 14 destabilizing mutations. The results show that the ligand exchange equilibrium is controlled by the stability of the same foldon unit implicated before. In addition, the results obtained confirm the epsilon-amino group of Lys79 and Lys73 as the alkaline replacement ligands and bear on the search for a triggering group. PMID:16287119

  4. The 29 DNA Polymerase: Protein-Primer Structure Suggests a Model of the Initiation to Elongation Transition

    SciTech Connect

    Kamtekar,S.; Berman, A.; Wang, J.; Lazaro, J.; Vega, M.; Blanco, L.; Salas, M.; Steitz, T.

    2006-01-01

    The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage {phi}29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 Angstroms resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

  5. Transitive Homology-Guided Structural Studies Lead to Discovery of Cro Proteins With 40% Sequence Identify But Different Folds

    SciTech Connect

    Roessler, C.G.; Hall, B.M.; Anderson, W.J.; Ingram, W.M.; Roberts, S.A.; Montfort, W.R.; Cordes, M.H.J.

    2009-05-27

    Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a 'stepping-stone' method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and {lambda}. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and {lambda}. The domains show 40% sequence identity but differ by switching of {alpha}-helix to {beta}-sheet in a C-terminal region spanning {approx}25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.

  6. TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome.

    PubMed

    Lambacher, Nils J; Bruel, Ange-Line; van Dam, Teunis J P; Szymańska, Katarzyna; Slaats, Gisela G; Kuhns, Stefanie; McManus, Gavin J; Kennedy, Julie E; Gaff, Karl; Wu, Ka Man; van der Lee, Robin; Burglen, Lydie; Doummar, Diane; Rivière, Jean-Baptiste; Faivre, Laurence; Attié-Bitach, Tania; Saunier, Sophie; Curd, Alistair; Peckham, Michelle; Giles, Rachel H; Johnson, Colin A; Huynen, Martijn A; Thauvin-Robinet, Christel; Blacque, Oliver E

    2016-01-01

    The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture. PMID:26595381

  7. Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes.

    PubMed

    Tubasum, Sumera; Torbjörnsson, Magne; Yadav, Dheerendra; Camacho, Rafael; Söderlind, Gustaf; Scheblykin, Ivan G; Pullerits, Tõnu

    2016-02-01

    Protein is a flexible material with broad distribution of conformations forming an energy landscape of quasi-stationary states. Disentangling the system dynamics along this landscape is the key for understanding the functioning of the protein. Here we studied a photosynthetic antenna pigment-protein complex LH2 with single molecule two-dimensional polarization imaging. Modeling based on the Redfield relaxation theory well describes the observed polarization properties of LH2 fluorescence and fluorescence excitation, strongly suggesting that at 77 K the conformational subspace of the LH2 is limited to about three configurations with relatively frequent switching among each other. At room temperature the next level of fluctuations determines the conformational dynamics. The results support the multitier model of the energy landscape of proteins and demonstrate the potential of the method for the studies of structural dynamics in proteins. PMID:26741912

  8. Dynamical phase transitions reveal amyloid-like states on protein folding landscapes.

    PubMed

    Weber, Jeffrey K; Jack, Robert L; Schwantes, Christian R; Pande, Vijay S

    2014-08-19

    Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein's dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation. PMID:25140433

  9. Backbone dynamics of barstar: a (15)N NMR relaxation study.

    PubMed

    Sahu, S C; Bhuyan, A K; Majumdar, A; Udgaonkar, J B

    2000-12-01

    Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2

  10. The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle-luteal transition.

    PubMed

    Joseph, Chitra; Hunter, Morag G; Sinclair, Kevin D; Robinson, Robert S

    2012-09-01

    The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development. PMID:22733805

  11. Carbon backbone topology of the metabolome of a cell.

    PubMed

    Bingol, Kerem; Zhang, Fengli; Bruschweiler-Li, Lei; Brüschweiler, Rafael

    2012-05-30

    The complex metabolic makeup of a biological system, such as a cell, is a key determinant of its biological state providing unique insights into its function. Here we characterize the metabolome of a cell by a novel homonuclear (13)C 2D NMR approach applied to a nonfractionated uniformly (13)C-enriched lysate of E. coli cells and determine de novo their carbon backbone topologies that constitute the "topolome". A protocol was developed, which first identifies traces in a constant-time (13)C-(13)C TOCSY NMR spectrum that are unique for individual mixture components and then assembles for each trace the corresponding carbon-bond topology network by consensus clustering. This led to the determination of 112 topologies of unique metabolites from a single sample. The topolome is dominated by carbon topologies of carbohydrates (34.8%) and amino acids (45.5%) that can constitute building blocks of more complex structures. PMID:22540339

  12. Efficient synthesis of longer Aβ peptides via removable backbone modification.

    PubMed

    Zuo, Chao; Tang, Shan; Si, Yan-Yan; Wang, Zhipeng A; Tian, Chang-Lin; Zheng, Ji-Shen

    2016-06-14

    Longer amyloid-beta (Aβ) peptides (43 to 49 amino acids) play essential roles in the pathology of Alzheimer's disease (AD). The difficulty in the preparation of longer Aβ peptides is still an obstacle to elucidate their roles in AD. Herein we report a robust and efficient strategy for the chemical synthesis of longer Aβ peptides (Aβ48 and Aβ49). A key feature of this method is the installation of removable Arg4-tagged backbone modification groups into the hydrophobic region of Aβ. This modification can improve the handling properties of the purification, ligation and mass characterization of longer Aβ peptides. The practicability of the new method has been demonstrated by the successful synthesis of Aβ48 and Aβ49 peptides. PMID:27188564

  13. Transforming plastic surfaces with electrophilic backbones from hydrophobic to hydrophilic.

    PubMed

    Kim, Samuel; Bowen, Raffick A R; Zare, Richard N

    2015-01-28

    We demonstrate a simple nonaqueous reaction scheme for transforming the surface of plastics from hydrophobic to hydrophilic. The chemical modification is achieved by base-catalyzed trans-esterification with polyols. It is permanent, does not release contaminants, and causes no optical or mechanical distortion of the plastic. We present contact angle measurements to show successful modification of several types of plastics including poly(ethylene terephthalate) (PET) and polycarbonate (PC). Its applicability to blood analysis is explored using chemically modified PET blood collection tubes and found to be quite satisfactory. We expect this approach will reduce the cost of manufacturing plastic devices with optimized wettability and can be generalized to other types of plastic materials having an electrophilic linkage as its backbone. PMID:25565370

  14. The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans

    PubMed Central

    2010-01-01

    Background Comparative genomics of the early diverging metazoan lineages and of their unicellular sister-groups opens new window to reconstructing the genetic changes which preceded or accompanied the evolution of multicellular body plans. A recent analysis found that the genome of the nerve-less sponges encodes the homologues of most vertebrate post-synaptic proteins. In vertebrate excitatory synapses, these proteins assemble to form the post-synaptic density, a complex molecular platform linking membrane receptors, components of their signalling pathways, and the cytoskeleton. Newly available genomes from Monosiga brevicollis (a member of Choanoflagellata, the closest unicellular relatives of animals) and Trichoplax adhaerens (a member of Placozoa: besides sponges, the only nerve-less metazoans) offer an opportunity to refine our understanding of post-synaptic protein evolution. Results Searches for orthologous proteins and reconstruction of gene gains/losses based on the taxon phylogeny indicate that post-synaptic proteins originated in two main steps. The backbone scaffold proteins (Shank, Homer, DLG) and some of their partners were acquired in a unicellular ancestor of choanoflagellates and metazoans. A substantial additional set appeared in an exclusive ancestor of the Metazoa. The placozoan genome contains most post-synaptic genes but lacks some of them. Notably, the master-scaffold protein Shank might have been lost secondarily in the placozoan lineage. Conclusions The time of origination of most post-synaptic proteins was not concomitant with the acquisition of synapses or neural-like cells. The backbone of the scaffold emerged in a unicellular context and was probably not involved in cell-cell communication. Based on the reconstructed protein composition and potential interactions, its ancestral function could have been to link calcium signalling and cytoskeleton regulation. The complex later became integrated into the evolving synapse through the

  15. SOP-GPU: influence of solvent-induced hydrodynamic interactions on dynamic structural transitions in protein assemblies.

    PubMed

    Alekseenko, Andrey; Kononova, Olga; Kholodov, Yaroslav; Marx, Kenneth A; Barsegov, Valeri

    2016-06-30

    Hydrodynamic interactions (HI) are incorporated into Langevin dynamics of the Cα -based protein model using the Truncated Expansion approximation (TEA) to the Rotne-Prager-Yamakawa diffusion tensor. Computational performance of the obtained GPU realization demonstrates the model's capability for describing protein systems of varying complexity (10(2) -10(5) residues), including biological particles (filaments, virus shells). Comparison of numerical accuracy of the TEA versus exact description of HI reveals similar results for the kinetics and thermodynamics of protein unfolding. The HI speed up and couple biomolecular transitions through cross-communication among protein domains, which result in more collective displacements of structure elements governed by more deterministic (less variable) dynamics. The force-extension/deformation spectra from nanomanipulations in silico exhibit sharper force signals that match well the experimental profiles. Hence, biomolecular simulations without HI overestimate the role of tension/stress fluctuations. Our findings establish the importance of incorporating implicit water-mediated many-body effects into theoretical modeling of dynamic processes involving biomolecules. © 2016 Wiley Periodicals, Inc. PMID:27015749

  16. Nuclear transition protein 1 from ram elongating spermatids. Mass spectrometric characterization, primary structure and phosphorylation sites of two variants.

    PubMed

    Chirat, F; Martinage, A; Briand, G; Kouach, M; Van Dorsselaer, A; Loir, M; Sautière, P

    1991-05-23

    The ram transition protein 1 (TP1) is present in spermatid cell nuclei in the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its primary structure was determined by automated Edman degradation of S-carboxamidomethylated protein and of peptides generated by cleavage with thermolysin and endoproteinase Lys-C. The ram TP1 is a small basic protein of 54 residues and structurally very close to other mammalian TP1. The mass spectrometric data obtained from the protein and its fragments reveal that ram TP1 is indeed a mixture (approximately 5:1) of two structural variants (Mr 6346 and 6300). These variants differ only by the nature of the residue at position 27 (Cys in the major variant and Gly in the minor variant). The study of phosphorylation sites has shown that four different serine residues could be phosphorylated in the monophosphorylated TP1, at positions 8, 35, 36 or 39. From previous physical studies, it has been postulated that the Tyr32 surrounded by two highly conserved basic clusters was responsible for the destabilization of chromatin by intercalation of its phenol ring between the bases of double-stranded DNA. The presence of three phosphorylatable serine residues in the very conserved sequence 29-42 is another argument for the involvement of this region in the interaction with DNA. PMID:2040274

  17. Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding

    PubMed Central

    Stigler, Johannes; Rief, Matthias

    2015-01-01

    Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins. PMID:26200872

  18. Investigating the roles of dimensionality in the helix-coil transition in random walk protein models using the method of Lee-Yang zeros

    NASA Astrophysics Data System (ADS)

    Linhananta, Apichart

    2004-03-01

    Recent computer simulations of coarse-grained (J.P. Kemp and Z.Y. Chen, 1998, Phys. Rev. Lett. 81, 3880) and all-atom protein models (M. Takano et. al., 2002, J. Chem. Phys. 116, 2219) have demonstrated that the helix-coil transition is a first-order-like transition. This contradicts the classical Ising-based one-dimensional Zimm-Bragg theory in which no phase transition occurs. It was conjectured that the discrepancy is due to long-range interactions and the fact that real protein systems are not one dimensional. The effects of long-range interactions have been investigated by minimalist models (N.A. Alves and U.H.E. Hansmann, 2000, Phys. Rev. Lett. 84, 1836; J.P. Kemp, U.H.E. Hansmann, and Z.Y. Chen, 2000, Eur. Phys. J. B, 15, 371), which suggests the universality of the helix-coil transition. This paper examines how conformation entropy of proteins in two or three dimensions drives the helix-coil transition. This is done by constructing two- or three-dimension self-avoiding random-walk models of proteins in which it is energetically favorable for the protein to assume linear (helical) configurations. The partition functions were determined exactly. Using the method of finite-size scaling and Lee-Yang zeros, it was confirmed that the phase transition is first order-like. The model is then extended to examine proteins that can form tertiary structures and non-native interactions. It was found that the partition-function zeros of these models differ significantly from those of the helix-coil models. The characterization of phase transitions in models of proteins and biopolymers by Lee-Yang zeros are discussed.

  19. Are all regions of folded proteins that undergo ligand-dependent order-disorder transitions targets for allosteric peptide mimetics?†

    PubMed Central

    Fenton, Aron W.

    2013-01-01

    Although the classical view of how proteins function relied on well folded structures, it is now recognized that the function of many proteins is dependent on being intrinsically disordered. The primary consideration in this work is the intermediate group of proteins that are overall well folded, but which contain small regions that undergo order/disorder transitions. In particular, the current focus is on those order/disorder transitions that are energetically coupled to ligand binding. As exemplified by the case of human liver pyruvate kinase (hL-PYK), peptides that mimic the sequence of the order/disorder region can be used as allosteric regulators of the enzyme. Based on this example and others reported in the literature, we propose that a similar use of peptides that mimic protein regions that experience ligand-dependent order-disorder transitions can be a generalized initiation point for the development of allosteric drugs. PMID:23520021

  20. Phase transitions of multivalent proteins can promote clustering of membrane receptors

    PubMed Central

    Banjade, Sudeep; Rosen, Michael K

    2014-01-01

    Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones. DOI: http://dx.doi.org/10.7554/eLife.04123.001 PMID:25321392

  1. Single-Molecule Analysis of Protein Large-Amplitude Conformational Transitions

    NASA Astrophysics Data System (ADS)

    Yang, Haw

    2011-03-01

    Proteins have evolved to harness thermal fluctuations, rather than frustrated by them, to carry out chemical transformations and mechanical work. What are, then, the operation and design principles of protein machines? To frame the problem in a tractable way, several basic questions have been formulated to guide the experimental design: (a) How many conformational states can a protein sample on the functionally important timescale? (b) What are the inter-conversion rates between states? (c) How do ligand binding or interactions with other proteins modulate the motions? (d) What are the structural basis of flexibility and its underlying molecular mechanics? Guided by this framework, we have studied protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis and a potential drug target) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells). These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. Integrating these microscopic dynamics into macroscopic kinetics allows us to place the experimentally measured free-energy landscape in the context of enzymatic turnovers.

  2. The evolutionary transition from monomeric to oligomeric proteins: tools, the environment, hypotheses.

    PubMed

    D'Alessio, G

    1999-01-01

    Recently, renewed interest in the evolution of oligomeric proteins has seen new approaches explored and new hypotheses proposed. The model systems chosen are generally made up of pairs of homologous proteins, each composed of a monomer and a dimeric counterpart, but the question has been also approached by comparing statistically significant structural patterns in sets of monomeric and oligomeric proteins. Here the tools of genetics and chemistry potentially available to the evolution of oligomeric proteins are discussed, as well as the possible effects of environments on the early attempts to oligomerization. Traces of an ancestral monomeric status of oligomers may be detected in the significant presence of polar and charged residues at intersubunit interfaces, and by the recognition that, besides the hydrophobic effect, a 'hydrophilic' effect has also had a role in the construction of these interfaces. The traditional 'mutation' model is described and found to be based on a hierarchy of mutations, crowned by a 'primary' mutation, one that could prime oligomerization by irreversibly altering the structure of an ancestral monomer. The mechanism of oligomerization based on the exchange or 'swap' of structural elements between monomers is discussed. The possibility is also discussed that the main steps in the folding pathway of an oligomeric protein reiterate the main steps in its evolution. PMID:10581971

  3. Thin Films Formed from Conjugated Polymers with Ionic, Water-Soluble Backbones.

    PubMed

    Voortman, Thomas P; Chiechi, Ryan C

    2015-12-30

    This paper compares the morphologies of films of conjugated polymers in which the backbone (main chain) and pendant groups are varied between ionic/hydrophilic and aliphatic/hydrophobic. We observe that conjugated polymers in which the pendant groups and backbone are matched, either ionic-ionic or hydrophobic-hydrophobic, form smooth, structured, homogeneous films from water (ionic) or tetrahydrofuran (hydrophobic). Mismatched conjugated polymers, by contrast, form inhomogeneous films with rough topologies. The polymers with ionic backbone chains are conjugated polyions (conjugated polymers with closed-shell charges in the backbone), which are semiconducting materials with tunable bad-gaps, not unlike uncharged conjugated polymers. PMID:25723354

  4. A high resolution electro-optical approach for investigating transition of soluble proteins to integral membrane proteins probed by colicin A.

    PubMed

    Honigmann, Alf; Pulagam, Lakshmi Padmavathi; Sippach, Michael; Bartsch, Philipp; Steinhoff, Heinz-Jürgen; Wagner, Richard

    2012-10-19

    The transition from water soluble state to an integral membrane protein state is a crucial step in the formation of the active form of many pore-forming or receptor proteins. Albeit this, high resolution techniques which allow assay of protein membrane binding and concomitant development of the final active form in the membrane await further development. Here, we describe a horizontal artificial bilayers setup allowing for simultaneous electrical and optical measurements at a single molecule level. We use the membrane binding and subsequent channel formation of colicin A (ColA) a water soluble bacteriocin secreted by some strains of Escherichia coli to demonstrate the potential of the combined electro-optical technique. Our results expand the knowledge on ColA molecular details which show that active ColA is monomeric; membrane binding is pH but not membrane-potential (Δϕ) dependent. ColA is at Δϕ=0 permeable for molecules ≥1 nm. Although ColA exhibits low ion conductance it facilitates permeation of large molecules. Our electro-optical recordings reveal ColA monomeric state and the chimeric character of its pore. PMID:23000162

  5. Novel Variants of AbaR Resistance Islands with a Common Backbone in Acinetobacter baumannii Isolates of European Clone II

    PubMed Central

    Povilonis, Justas; Sužiedėlienė, Edita

    2012-01-01

    In this study, the genetic organization of three novel genomic antibiotic resistance islands (AbaRs) in Acinetobacter baumannii isolates belonging to group of European clone II (EC II) comM integrated sequences of 18-, 21-, and 23-kb resistance islands were determined. These resistance islands carry the backbone of AbaR-type transposon structures, which are composed of the transposition module coding for potential transposition proteins and other genes coding for the intact universal stress protein (uspA), sulfate permease (sul), and proteins of unknown function. The antibiotic resistance genes strA, strB, tetB, and tetR and insertion sequence CR2 element were found to be inserted into the AbaR transposons. GenBank homology searches indicated that they are closely related to the AbaR sequences found integrated in comM in strains of EC II (A. baumannii strains 1656-2 and TCDC-AB0715) and AbaR4 integrated in another location of A. baumannii AB0057 (EC I). All of the AbaRs showed structural similarity to the previously described AbaR4 island and share a 12,008-bp backbone. AbaRs contain Tn1213, Tn2006, and the multiple fragments which could be derived from transposons Tn3, Tn10, Tn21, Tn1000, Tn5393, and Tn6020, the insertion sequences IS26, ISAba1, ISAba14, and ISCR2, and the class 1 integron. Moreover, chromosomal DNA was inserted into distinct regions of the AbaR backbone. Sequence analysis suggested that the AbaR-type transposons have evolved through insertions, deletions, and homologous recombination. AbaR islands, sharing the core structure similar to AbaR4, appeared to be distributed in isolates of EC I and EC II via integration into distinct genomic sites, i.e., pho and comM, respectively. PMID:22290980

  6. Eukaryotic Evolutionary Transitions Are Associated with Extreme Codon Bias in Functionally-Related Proteins

    PubMed Central

    Hudson, Nicholas J.; Gu, Quan; Nagaraj, Shivashankar H.; Ding, Yong-Sheng; Dalrymple, Brian P.; Reverter, Antonio

    2011-01-01

    Codon bias in the genome of an organism influences its phenome by changing the speed and efficiency of mRNA translation and hence protein abundance. We hypothesized that differences in codon bias, either between-species differences in orthologous genes, or within-species differences between genes, may play an evolutionary role. To explore this hypothesis, we compared the genome-wide codon bias in six species that occupy vital positions in the Eukaryotic Tree of Life. We acquired the entire protein coding sequences for these organisms, computed the codon bias for all genes in each organism and explored the output for relationships between codon bias and protein function, both within- and between-lineages. We discovered five notable coordinated patterns, with extreme codon bias most pronounced in traits considered highly characteristic of a given lineage. Firstly, the Homo sapiens genome had stronger codon bias for DNA-binding transcription factors than the Saccharomyces cerevisiae genome, whereas the opposite was true for ribosomal proteins – perhaps underscoring transcriptional regulation in the origin of complexity. Secondly, both mammalian species examined possessed extreme codon bias in genes relating to hair – a tissue unique to mammals. Thirdly, Arabidopsis thaliana showed extreme codon bias in genes implicated in cell wall formation and chloroplast function – which are unique to plants. Fourthly, Gallus gallus possessed strong codon bias in a subset of genes encoding mitochondrial proteins – perhaps reflecting the enhanced bioenergetic efficiency in birds that co-evolved with flight. And lastly, the G. gallus genome had extreme codon bias for the Ciliary Neurotrophic Factor – which may help to explain their spontaneous recovery from deafness. We propose that extreme codon bias in groups of genes that encode functionally related proteins has a pathway-level energetic explanation. PMID:21966531

  7. Exocyclic groups in the minor groove influence the backbone conformation of DNA

    PubMed Central

    Wellenzohn, Bernd; Flader, Wolfgang; Winger, Rudolf H.; Hallbrucker, Andreas; Mayer, Erwin; Liedl, Klaus R.

    2001-01-01

    Exocyclic groups in the minor groove of DNA modulate the affinity and positioning of nucleic acids to the histone protein. The addition of exocyclic groups decreases the formation of this protein–DNA complex, while their removal increases nucleosome formation. On the other hand, recent theoretical results show a strong correlation between the BI/BII phosphate backbone conformation and the hydration of the grooves of the DNA. We performed a simulation of the d(CGCGAATTCGCG)2 Drew Dickerson dodecamer and one simulation of the d(CGCIAATTCGCG)2 dodecamer in order to investigate the influence of the exocyclic amino group of guanine. The removal of the amino group introduces a higher intrinsic flexibility to DNA supporting the suggestions that make the enhanced flexibility responsible for the enlarged histone complexation affinity. This effect is attributed to changes in the destacking interactions of both strands of the DNA. The differences in the hydration of the minor groove could be the explanation of this flexibility. The changed hydration of the minor groove also leads to a different BI/BII substate pattern. Due to the fact that the histone preferentially builds contacts with the backbone of the DNA, we propose an influence of these BI/BII changes on the nucleosome formation process. Thus, we provide an additional explanation for the enhanced affinity to the histone due to removal of exocyclic groups. In terms of BI/BII we are also able to explain how minor groove binding ligands could affect the nucleosome assembly without disrupting the structure of DNA. PMID:11812834

  8. An avian live attenuated master backbone for potential use in epidemic and pandemic influenza vaccines

    PubMed Central

    Hickman, Danielle; Hossain, Md Jaber; Song, Haichen; Araya, Yonas; Solórzano, Alicia; Perez, Daniel R.

    2008-01-01

    The unprecedented emergence in Asia of multiple avian influenza virus (AIV) subtypes with a broad host range poses a major challenge in the design of vaccination strategies that are both effective and available in a timely manner. The present study focused on the protective effects of a genetically modified AIV as a source for the preparation of vaccines for epidemic and pandemic influenza. It has previously been demonstrated that a live attenuated AIV based on the internal backbone of influenza A/Guinea fowl/Hong Kong/WF10/99 (H9N2), called WF10att, is effective at protecting poultry species against low- and high-pathogenicity influenza strains. More importantly, this live attenuated virus provided effective protection when administered in ovo. In order to characterize the WF10att backbone further for use in epidemic and pandemic influenza vaccines, this study evaluated its protective effects in mice. Intranasal inoculation of modified attenuated viruses in mice provided adequate protective immunity against homologous lethal challenges with both the wild-type influenza A/WSN/33 (H1N1) and A/Vietnam/1203/04 (H5N1) viruses. Adequate heterotypic immunity was also observed in mice vaccinated with modified attenuated viruses carrying H7N2 surface proteins. The results presented in this report suggest that the internal genes of a genetically modified AIV confer similar protection in a mouse model and thus could be used as a master donor strain for the generation of live attenuated vaccines for epidemic and pandemic influenza. PMID:18931063

  9. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor.

    PubMed

    Isogai, Shin; Deupi, Xavier; Opitz, Christian; Heydenreich, Franziska M; Tsai, Ching-Ju; Brueckner, Florian; Schertler, Gebhard F X; Veprintsev, Dmitry B; Grzesiek, Stephan

    2016-02-11

    G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental

  10. Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System

    PubMed Central

    2016-01-01

    Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking–MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry–MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation. PMID:27504491

  11. Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein.

    PubMed

    Weise, Christoph F; Login, Frédéric H; Ho, Oanh; Gröbner, Gerhard; Wolf-Watz, Hans; Wolf-Watz, Magnus

    2014-10-21

    The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia. PMID:25418176

  12. Bisphenol A modulates colorectal cancer protein profile and promotes the metastasis via induction of epithelial to mesenchymal transitions.

    PubMed

    Chen, Zhuo-Jia; Yang, Xiang-Ling; Liu, Hao; Wei, Wei; Zhang, Kun-Shui; Huang, Hong-Bin; Giesy, John P; Liu, Huan-Liang; Du, Jun; Wang, Hong-Sheng

    2015-08-01

    More and more evidences indicate that endocrine disruptor chemicals such as bisphenol A (BPA) can act as carcinogens and enhance susceptibility to tumorigenesis. Although the gut is in direct contact with orally ingested BPA, effects of BPA on occurrence and development of colorectal cancer remain an unexplored endpoint. Colorectal cancer SW480 cells treated with nanomolar (10(-8) M) or greater (10(-5) M) concentrations of BPA were compared with responses of a control group. Proteomic study revealed that more than 56 proteins were modulated following exposure to BPA, which are relevant to structure, motility and proliferation of cells, production of ATP, oxidative stress, and protein metabolism. Further studies revealed that BPA increased migration and invasion and triggered transformations from epithelial to mesenchymal transitions (EMTs) of colorectal cancer cells, which was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin with a concomitant decrease of E-cadherin. Accordingly, BPA treatment increased the expression of transcription factor Snail. Furthermore, signal AKT/GSK-3β-mediated stabilization of Snail is involved during BPA-induced EMT of colon cancer cells. Our study first demonstrated that the xenoestrogen BPA at nanomolar and greater concentrations modulates the protein profiles and promotes the metastasis of colorectal cancer cells via induction of EMT. PMID:25119493

  13. Chromatinized protein kinase C-θ directly regulates inducible genes in epithelial to mesenchymal transition and breast cancer stem cells.

    PubMed

    Zafar, Anjum; Wu, Fan; Hardy, Kristine; Li, Jasmine; Tu, Wen Juan; McCuaig, Robert; Harris, Janelle; Khanna, Kum Kum; Attema, Joanne; Gregory, Philip A; Goodall, Gregory J; Harrington, Kirsti; Dahlstrom, Jane E; Boulding, Tara; Madden, Rebecca; Tan, Abel; Milburn, Peter J; Rao, Sudha

    2014-08-01

    Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. PMID:24891615

  14. Chromatinized Protein Kinase C-θ Directly Regulates Inducible Genes in Epithelial to Mesenchymal Transition and Breast Cancer Stem Cells

    PubMed Central

    Zafar, Anjum; Wu, Fan; Hardy, Kristine; Li, Jasmine; Tu, Wen Juan; McCuaig, Robert; Harris, Janelle; Khanna, Kum Kum; Attema, Joanne; Gregory, Philip A.; Goodall, Gregory J.; Harrington, Kirsti; Dahlstrom, Jane E.; Boulding, Tara; Madden, Rebecca; Tan, Abel; Milburn, Peter J.

    2014-01-01

    Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. PMID:24891615

  15. Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

    PubMed Central

    Chotewutmontri, Prakitchai; Bruce, Barry D.

    2015-01-01

    Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

  16. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

    PubMed Central

    Lier, Johanna Maria; Burmühl, Stephan; Struchtrup, Andreas; Deutschmann, Kathleen; Vetter, Maik; Leu, Tristan; Reeg, Sandra; Grune, Tilman; Rüther, Ulrich

    2015-01-01

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. PMID:26150391

  17. High-Resolution Mapping of the Folding Transition State of a WW Domain.

    PubMed

    Dave, Kapil; Jäger, Marcus; Nguyen, Houbi; Kelly, Jeffery W; Gruebele, Martin

    2016-04-24

    Fast-folding WW domains are among the best-characterized systems for comparing experiments and simulations of protein folding. Recent microsecond-resolution experiments and long duration (totaling milliseconds) single-trajectory modeling have shown that even mechanistic changes in folding kinetics due to mutation can now be analyzed. Thus, a comprehensive set of experimental data would be helpful to benchmark the predictions made by simulations. Here, we use T-jump relaxation in conjunction with protein engineering and report mutational Φ-values (ΦM) as indicators for folding transition-state structure of 65 side chain, 7 backbone hydrogen bond, and 6 deletion and /or insertion mutants within loop 1 of the 34-residue hPin1 WW domain. Forty-five cross-validated consensus mutants could be identified that provide structural constraints for transition-state structure within all substructures of the WW domain fold (hydrophobic core, loop 1, loop 2, β-sheet). We probe the robustness of the two hydrophobic clusters in the folding transition state, discuss how local backbone disorder in the native-state can lead to non-classical ΦM-values (ΦM > 1) in the rate-determining loop 1 substructure, and conclusively identify mutations and positions along the sequence that perturb the folding mechanism from loop 1-limited toward loop 2-limited folding. PMID:26880334

  18. Live-attenuated influenza A virus vaccines using a B virus backbone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The currently FDA-licensed live attenuated influenza virus vaccine contains a trivalent mixture of types A (H1N1 and H3N2) and B vaccine viruses. The two A virus vaccines have the backbone of a cold-adapted influenza A virus and the B virus vaccine has the six backbone segments derived from a cold-...

  19. Quantitative Analysis of PMLA Nanoconjugate Components after Backbone Cleavage

    PubMed Central

    Ding, Hui; Patil, Rameshwar; Portilla-Arias, Jose; Black, Keith L.; Ljubimova, Julia Y.; Holler, Eggehard

    2015-01-01

    Multifunctional polymer nanoconjugates containing multiple components show great promise in cancer therapy, but in most cases complete analysis of each component is difficult. Polymalic acid (PMLA) based nanoconjugates have demonstrated successful brain and breast cancer treatment. They consist of multiple components including targeting antibodies, Morpholino antisense oligonucleotides (AONs), and endosome escape moieties. The component analysis of PMLA nanoconjugates is extremely difficult using conventional spectrometry and HPLC method. Taking advantage of the nature of polyester of PMLA, which can be cleaved by ammonium hydroxide, we describe a method to analyze the content of antibody and AON within nanoconjugates simultaneously using SEC-HPLC by selectively cleaving the PMLA backbone. The selected cleavage conditions only degrade PMLA without affecting the integrity and biological activity of the antibody. Although the amount of antibody could also be determined using the bicinchoninic acid (BCA) method, our selective cleavage method gives more reliable results and is more powerful. Our approach provides a new direction for the component analysis of polymer nanoconjugates and nanoparticles. PMID:25894227

  20. Backbones of evolutionary history test biodiversity theory for microbes

    PubMed Central

    O’Dwyer, James P.; Kembel, Steven W.; Sharpton, Thomas J.

    2015-01-01

    Identifying the ecological and evolutionary mechanisms that determine biological diversity is a central question in ecology. In microbial ecology, phylogenetic diversity is an increasingly common and relevant means of quantifying community diversity, particularly given the challenges in defining unambiguous species units from environmental sequence data. We explore patterns of phylogenetic diversity across multiple bacterial communities drawn from different habitats and compare these data to evolutionary trees generated using theoretical models of biodiversity. We have two central findings. First, although on finer scales the empirical trees are highly idiosyncratic, on coarse scales the backbone of these trees is simple and robust, consistent across habitats, and displays bursts of diversification dotted throughout. Second, we find that these data demonstrate a clear departure from the predictions of standard neutral theories of biodiversity and that an alternative family of generalized models provides a qualitatively better description. Together, these results lay the groundwork for a theoretical framework to connect ecological mechanisms to observed phylogenetic patterns in microbial communities. PMID:26106159

  1. Solid state radiation chemistry of the DNA backbone

    SciTech Connect

    Bernhard, W.A.

    1989-09-01

    The long term goal of this program is to determine the fundamental rules needed to predict the type and yield of damage produced in DNA due to direct effects of ionizing radiation. The focus is on damage to the sugar-phosphate backbone, damage that would lead to strand breaks. Model systems have been chosen that permit various aspects of this problem to be investigated. The emphasis will be on single crystals of monosaccharides, nucleosides, and nucleotides but will also include some powder work on polynucleotides. In these model systems, free radical products and reactions are observed by electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) techniques. The information thus gained is used in constructing rules that predict what primary free radicals are formed in single crystals of model compounds and the reactions stemming from the primary radicals. The formulation of a set of rules that work in model systems will represent a major advance toward formulating a set of rules that predict the direct damage in DNA itself. In a broader context this program is part of the effort to understand and predict the effects of exposure to ionizing radiation received at low dose rates over long periods of time. Assessment of low dose effects requires a basic understanding of the action of radiation at the molecular level. By contributing to that basic understanding, this program will help solve the problems of risk assessment under low dose conditions. 5 refs., 3 figs.

  2. Backbone of complex networks of corporations: the flow of control.

    PubMed

    Glattfelder, J B; Battiston, S

    2009-09-01

    We present a methodology to extract the backbone of complex networks based on the weight and direction of links, as well as on nontopological properties of nodes. We show how the methodology can be applied in general to networks in which mass or energy is flowing along the links. In particular, the procedure enables us to address important questions in economics, namely, how control and wealth are structured and concentrated across national markets. We report on the first cross-country investigation of ownership networks, focusing on the stock markets of 48 countries around the world. On the one hand, our analysis confirms results expected on the basis of the literature on corporate control, namely, that in Anglo-Saxon countries control tends to be dispersed among numerous shareholders. On the other hand, it also reveals that in the same countries, control is found to be highly concentrated at the global level, namely, lying in the hands of very few important shareholders. Interestingly, the exact opposite is observed for European countries. These results have previously not been reported as they are not observable without the kind of network analysis developed here. PMID:19905177

  3. Backbone of complex networks of corporations: The flow of control

    NASA Astrophysics Data System (ADS)

    Glattfelder, J. B.; Battiston, S.

    2009-09-01

    We present a methodology to extract the backbone of complex networks based on the weight and direction of links, as well as on nontopological properties of nodes. We show how the methodology can be applied in general to networks in which mass or energy is flowing along the links. In particular, the procedure enables us to address important questions in economics, namely, how control and wealth are structured and concentrated across national markets. We report on the first cross-country investigation of ownership networks, focusing on the stock markets of 48 countries around the world. On the one hand, our analysis confirms results expected on the basis of the literature on corporate control, namely, that in Anglo-Saxon countries control tends to be dispersed among numerous shareholders. On the other hand, it also reveals that in the same countries, control is found to be highly concentrated at the global level, namely, lying in the hands of very few important shareholders. Interestingly, the exact opposite is observed for European countries. These results have previously not been reported as they are not observable without the kind of network analysis developed here.

  4. Hofmeister effects in protein unfolding kinetics: estimation of changes in surface area upon formation of the transition state.

    PubMed

    López-Arenas, Leticia; Solís-Mendiola, Silvia; Padilla-Zúñiga, Jaqueline; Hernández-Arana, Andrés

    2006-07-01

    We studied the effect of three electrolytes (LiCl, Na(2)SO(4), GuHCl) on the unfolding reaction of chymopapain, a two-domain protein belonging in the papain family of cysteine proteinases. Due to methodological reasons, these studies were carried out at pH 1.5 where the protein unfolds following biphasic kinetics. We have observed the presence of two different effects of electrolyte concentration on the unfolding reactions. At low ionic strength, the ionic atmosphere brought about an increase in reaction rates, regardless of the type of ions being present; this effect is attributed to a general "electrostatic screening" of charge-charge interactions in the macromolecule. At high ionic strength, each electrolyte exerted a distinctively different effect: both rate constants were largely increased by GuHCl (a well-known protein denaturant), but only slightly by LiCl; in contrast, Na(2)SO(4) (a good precipitant) decreased the value of both unfolding rates. These ion-specific (Hofmeister) effects were further used to estimate changes in accessible surface area (DeltaASA) upon formation of the transition states (TS) for unfolding. Results obtained with LiCl and Na(2)SO(4), which we analyzed by means of a parameterization derived from published solubility data of amino acid derivatives, are consistent with DeltaASA increments (for each phase) of about 8.0% of the total theoretical DeltaASA for complete unfolding of the chymopapain molecule. Results in the presence of GuHCl, which were analyzed by using a previous parameterization of protein unfolding data, gave larger DeltaASAs of activation, equivalent to 13 and 16% of the total unfolding DeltaASA. PMID:16837256

  5. Nanosecond Motions in Proteins Impose Bounds on the Timescale Distributions of Local Dynamics

    PubMed Central

    Okan, Osman Burak; Atilgan, Ali Rana; Atilgan, Canan

    2009-01-01

    Abstract We elucidate the physics of protein dynamical transition via 10–100-ns molecular dynamics simulations at temperatures spanning 160–300 K. By tracking the energy fluctuations, we show that the protein dynamical transition is marked by a crossover from nonstationary to stationary processes that underlie the dynamics of protein motions. A two-timescale function captures the nonexponential character of backbone structural relaxations. One timescale is attributed to the collective segmental motions and the other to local relaxations. The former is well defined by a single-exponential, nanosecond decay, operative at all temperatures. The latter is described by a set of processes that display a distribution of timescales. Although their average remains on the picosecond timescale, the distribution is markedly contracted at the onset of the transition. It is shown that the collective motions impose bounds on timescales spanned by local dynamical processes. The nonstationary character below the transition implicates the presence of a collection of substates whose interactions are restricted. At these temperatures, a wide distribution of local-motion timescales, extending beyond that of nanoseconds, is observed. At physiological temperatures, local motions are confined to timescales faster than nanoseconds. This relatively narrow window makes possible the appearance of multiple channels for the backbone dynamics to operate. PMID:19804740

  6. The TRIM-NHL Protein LIN-41 and the OMA RNA-Binding Proteins Antagonistically Control the Prophase-to-Metaphase Transition and Growth of Caenorhabditis elegans Oocytes

    PubMed Central

    Spike, Caroline A.; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-01-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation. PMID:25261698

  7. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes.

    PubMed

    Spike, Caroline A; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-12-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation. PMID:25261698

  8. Expression patterns of cyclin D1 and related proteins regulating G1-S phase transition in uveal melanoma and retinoblastoma

    PubMed Central

    Coupland, S; Bechrakis, N; Schuler, A; Anagnostopoulos, I; Hummel, M; Bornfeld, N; Stein, H

    1998-01-01

    BACKGROUND/AIMS—A checkpoint mechanism in late G1, whose regulation via loss of retinoblastoma protein (pRB) or p16, or overexpression of cyclin D1 or cyclin dependent kinase 4 (CDK4), has been proposed to constitute a common pathway to malignancy. The aims of this study were (a) to compare markers of cell cycle G1-S phase transition in an intraocular tumour with known pRB deficiency (retinoblastoma) and compare it with one with an apparently functional pRB (uveal melanoma); (b) to determine if one of these markers may have a role in the pathogenesis of uveal melanoma; and (c) to determine if there is a difference in cell cycle marker expression following treatment of uveal melanoma and retinoblastoma.
METHODS—90 eyes were enucleated from 89 patients for retinoblastoma (n=24) or for choroidal or ciliary body melanoma (n=66). Conventional paraffin sections were assessed for cell type and degree of differentiation. Additional slides were investigated applying standard immunohistochemical methods with antibodies specific for cyclin D1 protein, pRB, p53, p21, p16, BCL-2, and MIB-1.
RESULTS—Cyclin D1 protein and pRB were negative in retinoblastoma using the applied antibodies. In contrast, cyclin D1 protein expression was observed in 65% of uveal melanomas; a positive correlation between cyclin D1 cell positivity and tumour cell type, location, growth fraction, as well as with pRB positivity was observed. p53, p21, and p16 could be demonstrated in both tumours. An inverse relation between p53 and p21 expression was demonstrated in most choroidal melanomas and in some retinoblastomas. Apart from a decrease in the growth fractions of the tumours as determined by MIB-1, a significant difference in the expression of G1-S phase transition markers in vital areas of uveal melanoma and retinoblastoma following treatment with radiotherapy and/or chemotherapy was not observed.
CONCLUSION—Retinoblastomas and uveal melanomas, two tumours of differing pRB status

  9. Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

    PubMed

    Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

    2016-03-01

    K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution. PMID:26894407

  10. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition

    PubMed Central

    Azmi, Asfar S.; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A.; Mohammad, Ramzi M.

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (–ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  11. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

    PubMed

    Azmi, Asfar S; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A; Mohammad, Ramzi M

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (-ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  12. Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

    PubMed Central

    Nott, Timothy J.; Petsalaki, Evangelia; Farber, Patrick; Jervis, Dylan; Fussner, Eden; Plochowietz, Anne; Craggs, Timothy D.; Bazett-Jones, David P.; Pawson, Tony; Forman-Kay, Julie D.; Baldwin, Andrew J.

    2015-01-01

    Summary Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles. PMID:25747659

  13. The HMG-box-containing proteins tHMG-1 and tHMG-2 interact during the histone-to-protamine transition in Drosophila spermatogenesis.

    PubMed

    Gärtner, Stefanie M K; Rothenbusch, Silke; Buxa, Melanie K; Theofel, Ina; Renkawitz, Rainer; Rathke, Christina; Renkawitz-Pohl, Renate

    2015-01-01

    Spermatogenesis is accompanied by a remarkable reorganization of the chromatin in post-meiotic stages, characterized by a near genome-wide displacement of histones by protamines and a transient expression of transition proteins. In Drosophila, the transition-protein-like protein Tpl94D contains an HMG-box domain and is expressed during chromatin reorganization. Here, we searched for additional HMG-box-containing proteins with a similar expression pattern. We identified two proteins specifically expressed in the testis, tHMG-1 and tHMG-2, whose expression levels were highest during the histone-to-protamine transition. Protein-protein interaction studies revealed that tHMG-1 and tHMG-2 form heterodimers in vivo. We demonstrated that Tpl94D, tHMG-1 and tHMG-2 localize to chromatin of the male germ line, with the most abundant levels observed during post-meiotic chromatin reorganization. Analysis of a tpl94D mutant showed that the C-terminal region of Tpl94D is dispensable for fertility. These data strongly suggested either that the truncated protein, which still contains the N-terminal HMG-box domain, is functional or that other proteins act in functional redundancy with Tpl94D during spermiogenesis. A thmg-1/thmg-2 null mutant also had no detectable specific phenotype, but hmgz, which encodes the major somatic HMG-box-containing protein HMGZ, was transcriptionally up-regulated. Our results showed that Drosophila spermatogenesis is characterized by continuous and overlapping expression of different HMG-box-containing proteins. We hypothesize that the mechanism of chromatin reorganization is a process highly secured by redundancies. PMID:25464903

  14. Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid

    PubMed Central

    Babbitt, Gregory A.; Alawad, Mohammed A.; Schulze, Katharina V.; Hudson, André O.

    2014-01-01

    While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an ‘accessory’ during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context. PMID:25200075

  15. Backbone and side-chain resonance assignment of the A147T polymorph of mouse TSPO in complex with a high-affinity radioligand.

    PubMed

    Jaremko, Mariusz; Jaremko, Łukasz; Giller, Karin; Becker, Stefan; Zweckstetter, Markus

    2016-04-01

    The integral polytopic membrane protein TSPO is the target for numerous endogenous and synthetic ligands. However, the affinity of many ligands is influenced by a common polymorphism in TSPO, in which an alanine at position 147 is replaced by threonine, thereby complicating the use of several radioligands for clinical diagnosis. In contrast, the best-characterized TSPO ligand (R)-PK11195 binds with similar affinity to both variants of mitochondrial TSPO (wild-type and A147T variant). Here we report the (1)H, (13)C, (15)N backbone and side-chain resonance assignment of the A147T polymorph of TSPO from Mus Musculus in complex with (R)-PK11195 in DPC detergent micelles. More than 90 % of all resonances were sequence-specifically assigned, demonstrating the ability to obtain high-quality spectral data for both the backbone and the side-chains of medically relevant integral membrane proteins. PMID:26364056

  16. Diversity of extracellular proteins during the transition from the 'proto-apicomplexan' alveolates to the apicomplexan obligate parasites.

    PubMed

    Templeton, Thomas J; Pain, Arnab

    2016-01-01

    The recent completion of high-coverage draft genome sequences for several alveolate protozoans - namely, the chromerids, Chromera velia and Vitrella brassicaformis; the perkinsid Perkinsus marinus; the apicomplexan, Gregarina niphandrodes, as well as high coverage transcriptome sequence information for several colpodellids, allows for new genome-scale comparisons across a rich landscape of apicomplexans and other alveolates. Genome annotations can now be used to help interpret fine ultrastructure and cell biology, and guide new studies to describe a variety of alveolate life strategies, such as symbiosis or free living, predation, and obligate intracellular parasitism, as well to provide foundations to dissect the evolutionary transitions between these niches. This review focuses on the attempt to identify extracellular proteins which might mediate the physical interface of cell-cell interactions within the above life strategies, aided by annotation of the repertoires of predicted surface and secreted proteins encoded within alveolate genomes. In particular, we discuss what descriptions of the predicted extracellular proteomes reveal regarding a hypothetical last common ancestor of a pre-apicomplexan alveolate - guided by ultrastructure, life strategies and phylogenetic relationships - in an attempt to understand the evolution of obligate parasitism in apicomplexans. PMID:26585326

  17. Resistance issues with new nucleoside/nucleotide backbone options.

    PubMed

    Wainberg, Mark A; Turner, Dan

    2004-09-01

    The nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) remain an enduring feature of combination therapy. As NRTI/NtRTI options continue to expand, questions arise about how best to combine these agents to create effective dual NRTI/NtRTI backbones in antiretroviral regimens while avoiding treatment-emergent drug resistance. Clinicians must consider how NRTIs/NtRTIs such as tenofovir DF (TDF), abacavir (ABC), and emtricitabine (FTC), as well as new once-daily and coformulated NRTIs/NtRTIs, interact with older agents when combined in novel regimens and how sequencing the new NRTIs can preserve future treatment options. Resistance data from clinical trials have revealed important information on the patterns, prevalence, and effects of resistance seen among patients experiencing virologic failure. In recent years, the prevalence of some mutations such as M184V and Q151M has remained relatively constant, while the L74V mutation, the 69 insertions, and thymidine analogue mutations have decreased in prevalence. Other mutations such as K65R and Y115F, while still relatively uncommon, are increasing in prevalence. This increase may be due to the use of new treatment combinations that select for these mutations at a higher rate. Clinical trials suggest that new regimens containing TDF or ABC select for K65R and that this mutation is observed more frequently with TDF; in contrast, L74V is observed more frequently in ABC-containing regimens but is not commonly selected by TDF-containing regimens. Several lines of evidence are converging to suggest that the presence of zidovudine may decrease the risk of L74V and K65R in ABC- or TDF-containing regimens. This review summarizes the clinical implications of resistance profiles associated with new NRTI/NtRTI regimens in current use and in advanced clinical studies. PMID:15319668

  18. Conformation-specific spectroscopy of capped glutamine-containing peptides: role of a single glutamine residue on peptide backbone preferences.

    PubMed

    Walsh, Patrick S; Dean, Jacob C; McBurney, Carl; Kang, Hyuk; Gellman, Samuel H; Zwier, Timothy S

    2016-04-20

    The conformational preferences of a series of short, aromatic-capped, glutamine-containing peptides have been studied under jet-cooled conditions in the gas phase. This work seeks a bottom-up understanding of the role played by glutamine residues in directing peptide structures that lead to neurodegenerative diseases. Resonant ion-dip infrared (RIDIR) spectroscopy is used to record single-conformation infrared spectra in the NH stretch, amide I and amide II regions. Comparison of the experimental spectra with the predictions of calculations carried out at the DFT M05-2X/6-31+G(d) level of theory lead to firm assignments for the H-bonding architectures of a total of eight conformers of four molecules, including three in Z-Gln-OH, one in Z-Gln-NHMe, three in Ac-Gln-NHBn, and one in Ac-Ala-Gln-NHBn. The Gln side chain engages actively in forming H-bonds with nearest-neighbor amide groups, forming C8 H-bonds to the C-terminal side, C9 H-bonds to the N-terminal side, and an amide-stacked geometry, all with an extended (C5) peptide backbone about the Gln residue. The Gln side chain also stabilizes an inverse γ-turn in the peptide backbone by forming a pair of H-bonds that bridge the γ-turn and stabilize it. Finally, the entire conformer population of Ac-Ala-Gln-NHBn is funneled into a single structure that incorporates the peptide backbone in a type I β-turn, stabilized by the Gln side chain forming a C7 H-bond to the central amide group in the β-turn not otherwise involved in a hydrogen bond. This β-turn backbone structure is nearly identical to that observed in a series of X-(AQ)-Y β-turns in the protein data bank, demonstrating that the gas-phase structure is robust to perturbations imposed by the crystalline protein environment. PMID:27054830

  19. Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.

    PubMed

    Zhong, Qian; Zhou, Beiyun; Ann, David K; Minoo, Parviz; Liu, Yixin; Banfalvi, Agnes; Krishnaveni, Manda S; Dubourd, Mickael; Demaio, Lucas; Willis, Brigham C; Kim, Kwang-Jin; duBois, Roland M; Crandall, Edward D; Beers, Michael F; Borok, Zea

    2011-09-01

    Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis. PMID:21169555

  20. Epithelial-mesenchymal transition in human gastric cancer cell lines induced by TNF-α-inducing protein of Helicobacter pylori.

    PubMed

    Watanabe, Tatsuro; Takahashi, Atsushi; Suzuki, Kaori; Kurusu-Kanno, Miki; Yamaguchi, Kensei; Fujiki, Hirota; Suganuma, Masami

    2014-05-15

    Helicobacter pylori strains produce tumor necrosis factor-α (TNF-α)-inducing protein, Tipα as a carcinogenic factor in the gastric epithelium. Tipα acts as a homodimer with 38-kDa protein, whereas del-Tipα is an inactive monomer. H. pylori isolated from gastric cancer patients secreted large amounts of Tipα, which are incorporated into gastric cancer cells by directly binding to nucleolin on the cell surface, which is a receptor of Tipα. The binding complex induces expression of TNF-α and chemokine genes, and activates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells). To understand the mechanisms of Tipα in tumor progression, we looked at numerous effects of Tipα on human gastric cancer cell lines. Induction of cell migration and elongation was found to be mediated through the binding to surface nucleolin, which was inhibited by the nucleolin-targeted siRNAs. Tipα induced formation of filopodia in MKN-1 cells, suggesting invasive morphological changes. Tipα enhanced the phosphorylation of 11 cancer-related proteins in serine, threonine and tyrosine, indicating activation of MEK-ERK signal cascade. Although the downregulation of E-cadherin was not shown in MKN-1 cells, Tipα induced the expression of vimentin, a significant marker of the epithelial-mesenchymal transition (EMT). It is of great importance to note that Tipα reduced the Young's modulus of MKN-1 cells determined by atomic force microscopy: This shows lower cell stiffness and increased cell motility. The morphological changes induced in human gastric cancer cells by Tipα are significant phenotypes of EMT. This is the first report that Tipα is a new inducer of EMT, probably associated with tumor progression in human gastric carcinogenesis. PMID:24249671

  1. An instrument for time resolved monitoring of fast chemical transitions: Application to the kinetics of refolding of a globular protein

    NASA Astrophysics Data System (ADS)

    Ratner, Vladimir; Haas, Elisha

    1998-05-01

    The dynamics of kinetic intermediates of protein folding can be studied by time resolved measurements of nonradiative excitation energy transfer, in site-specific labeled protein derivatives, combined with fast mixing experiments. A new device based on the single pulse approach was developed. This experiment is performed over two time scales: the "chemical time scale" of the conformational changes (milliseconds), defined by the rates of changes of conformations in the sample, and the "spectroscopic time scale" (nanoseconds) defined by the lifetimes of the excited states of the fluorescent probes. The chemical process was synchronized by means of a fast mixing stopped flow device. The low cost laser used here is suitable for use with dyes with excitation wavelengths of 337 nm and higher. Up to 20 fluorescence decay curves per second, can be measured within a single stopped flow run. Each fluorescence decay curve is recorded within 250 ns or more. The time resolution (of the spectroscopic time scale) was 0.5 ns. The noise level is low enough to estimate distance distributions from energy transfer experiments, provided that the shortest changeable lifetime component of the fluorescence decay of the donor probes would not be lower than ˜4 ns. The amount of double labeled protein which should be used for each experiment in order to obtain a full data set, with time resolution of 10 ms during protein transition, is only fourfold more than the amount needed for a stopped flow study with steady state fluorescence monitoring. The results obtained for refolding of α-lactalbumin in the presence of 1,8-anilino-naphthalene sulfonic acid from the GuHCl induced denatured state, support the model in which the probe has two states. The first state, is characterized by a fluorescence lifetime of 14.2±0.5 ns and the second by a fluorescence lifetime of 0.5±0.4 ns or less. During refolding the dye is transferred from the first state to the second, at a rate that coincides with the

  2. Ab initio Study of Transition metal binding to the Prion Protein

    NASA Astrophysics Data System (ADS)

    Cox, Daniel L.; Singh, Rajiv R. P.; Pan, Jianping

    2004-03-01

    Fundamental understanding of the prion protein (PrP) is of critical public health importance in view of mad cow and chronic wasting diseases. In recent years, it has been shown that the normal form (PrP^c) binds copper^1), and the structure of the copper binding domain has been elaborated. Hypotheses about toxicity associated with binding of other metals (notably manganese) have been put forward, Accordingly, using the ab initio SIESTA density functional theory code^2), we calculated the binding energy E_B(M) of M-(PrP) complexes relative to initially uncomplexed M ions, with M=Cu,Ni,Zn,Mn and (PrP)^* the minimal binding domain. The binding energy trend is E_B(Ni)>E_B(Cu)>E_B(Zn)>E_B(Mn), consistent with recent experiments apart from the surprising stability of Ni. We will also present preliminary results for binding of initially complexed M ions. *-Supported by U.S. DOE, Office of Basic Energy Sciences, Division of Materials Research 1) G.S. Jackson et al., Proc. Nat. Acad. Sci. (USA) 98, 8531 (2001). 2) P. Ordejón, et al., Phys. Rev. B53, R10441 (1996); J.M. Soler et al., J. Phys. Cond. Matt. 14, 2745 (2002).

  3. Radiation-induced sol-gel transition of protein: Effects of metal ions on thermal property

    SciTech Connect

    Not Available

    1993-11-10

    The solid gelatin as the model protein, was irradiated with [sup 60]Co gamma rays in air at room temperature at dose rates of 6.0 x 10[sup 4] to 1.3 x 10[sup 5] rad/h. The irradiated solid gelatin was dissolved in distilled water or metal ion (Cu[sup 2][sup +] as CuSO[sub 4] of Fe[sup 2][sup +] as FeSO[sub 4]) solution at about 80 C. and held at 30 C. for 1 h. Then, the gelatin hydrosol was cooled at a rate of 0.2 C./min and the setting point measured. The heat energy required to associated cross-links of the gelatin hydrosol was calculated using the setting point given by the equation of Eldridge and Ferry. The changes in setting point of irradiated gelatin with and without metal ions (Cu[sup 2][sup +] and Fe[sup 2][sup +]) were studied with 3-10% gelatin and by the irradiation of 0, 10[sup 5], 10[sup 6], and 10[sup 7] rad.

  4. MCBT: Multi-Hop Cluster Based Stable Backbone Trees for Data Collection and Dissemination in WSNs.

    PubMed

    Shin, Inyoung; Kim, Moonseong; Mutka, Matt W; Choo, Hyunseung; Lee, Tae-Jin

    2009-01-01

    We propose a stable backbone tree construction algorithm using multi-hop clusters for wireless sensor networks (WSNs). The hierarchical cluster structure has advantages in data fusion and aggregation. Energy consumption can be decreased by managing nodes with cluster heads. Backbone nodes, which are responsible for performing and managing multi-hop communication, can reduce the communication overhead such as control traffic and minimize the number of active nodes. Previous backbone construction algorithms, such as Hierarchical Cluster-based Data Dissemination (HCDD) and Multicluster, Mobile, Multimedia radio network (MMM), consume energy quickly. They are designed without regard to appropriate factors such as residual energy and degree (the number of connections or edges to other nodes) of a node for WSNs. Thus, the network is quickly disconnected or has to reconstruct a backbone. We propose a distributed algorithm to create a stable backbone by selecting the nodes with higher energy or degree as the cluster heads. This increases the overall network lifetime. Moreover, the proposed method balances energy consumption by distributing the traffic load among nodes around the cluster head. In the simulation, the proposed scheme outperforms previous clustering schemes in terms of the average and the standard deviation of residual energy or degree of backbone nodes, the average residual energy of backbone nodes after disseminating the sensed data, and the network lifetime. PMID:22454570

  5. Nuclear magnetic resonance secondary shifts of a light-harvesting 2 complex reveal local backbone perturbations induced by its higher-order interactions.

    PubMed

    Pandit, Anjali; Wawrzyniak, Piotr K; van Gammeren, Adriaan J; Buda, Francesco; Ganapathy, Swapna; de Groot, Huub J M

    2010-01-26

    Protein nuclear magnetic resonance (NMR) secondary chemical shifts are widely used to predict the secondary structure, and in solid-state NMR, they are often the only unambiguous structural parameters available. However, the employed prediction methods are empirical in nature, relying on the assumption that secondary shifts are only affected by shielding effects of neighboring atoms. We analyzed the secondary shifts of a photosynthetic membrane protein with a high density of chromophores and very tight packing, the light-harvesting 2 (LH2) complex of Rhodopseudomonas acidophila. A relation was found between secondary shift anomalies and protein-protein or pigment-protein tertiary and quaternary contacts. For several residues, including the bacteriochlorophyll-coordinating histidines (alphaH31 and betaH30) and the phenylalanine alphaF41 that has strongly twisted C(b)-C(a)-C and C(a)-C-N conformations in the LH2 crystal structure, the perturbing effects on the backbone chemical shifts were tested by density functional theory (DFT) calculations. We propose that higher-order interactions in the tightly packed complex can induce localized perturbations of the backbone conformation and electronic structure, related to functional pigment-protein or protein-protein interactions. PMID:19954238

  6. Inositol-requiring protein 1 - X-box-binding protein 1 pathway promotes epithelial-mesenchymal transition via mediating snail expression in pulmonary fibrosis.

    PubMed

    Mo, Xiao-Ting; Zhou, Wen-Cheng; Cui, Wen-Hui; Li, De-Lin; Li, Liu-Cheng; Xu, Liang; Zhao, Ping; Gao, Jian

    2015-08-01

    Epithelial-mesenchymal transition (EMT) is a complex biological program during which cells loss epithelial phenotype and acquire mesenchymal features. EMT is thought to be involved in the pathogenesis of various fibrotic diseases including pulmonary fibrosis (PF). Recent studies suggest that endoplasmic reticulum (ER) stress is associated with EMT in the progression of PF. However, the exact mechanism is unclear. Here, we developed a PF model with bleomycin (BLM) administration in rats and conducted several simulation experiments in alveolar epithelial cell (AECs) RLE-6TN to unravel the role of inositol-requiring protein 1 (IRE1) - X-box-binding protein 1 (XBP1) signal pathway in ER stress-induced EMT in PF. First, we observed that ER stress was occurred in type II AECs accompanied by EMT in BLM-induced PF. Then we explored the role of IRE1-XBP1-snail pathway in transforming growth factor (TGF)-β1/tunicamycin (TM)-induced EMT. When TGF-β1/TM was treated on AECs, IRE1 and XBP1 were overexpressed, meanwhile, snail expression was upregulated accompanied with EMT. However, when IRE1 or XBP1 was knockdown, TGF-β1/TM-induced EMT were blocked while the expression of snail was inhibited. Then we silenced snail and found that TGF-β1/TM-induced EMT were also suppressed, but it had no effect on the up-regulated expression of IRE1 and XBP1. Thus, we concluded that IRE1-XBP1 pathway promotes EMT via mediating snail expression in PF. PMID:26065400

  7. Uganda's National Transmission Backbone Infrastructure Project: Technical Challenges and the Way Forward

    NASA Astrophysics Data System (ADS)

    Bulega, T.; Kyeyune, A.; Onek, P.; Sseguya, R.; Mbabazi, D.; Katwiremu, E.

    2011-10-01

    Several publications have identified technical challenges facing Uganda's National Transmission Backbone Infrastructure project. This research addresses the technical limitations of the National Transmission Backbone Infrastructure project, evaluates the goals of the project, and compares the results against the technical capability of the backbone. The findings of the study indicate a bandwidth deficit, which will be addressed by using dense wave division multiplexing repeaters, leasing bandwidth from private companies. Microwave links for redundancy, a Network Operation Center for operation and maintenance, and deployment of wireless interoperability for microwave access as a last-mile solution are also suggested.

  8. Spectroscopic Monitoring of Mechanical Forces during Protein Folding by using Molecular Force Probes.

    PubMed

    Stauch, Tim; Hoffmann, Marvin T; Dreuw, Andreas

    2016-05-18

    Detailed folding pathways of proteins are still largely unknown. Real-time monitoring of mechanical forces acting in proteins during structural transitions would provide deep insights into these highly complex processes. Here, we propose two molecular force probes that can be incorporated into the protein backbone to gain insight into the magnitude and direction of mechanical forces acting in proteins during natural folding and unfolding through their optical spectroscopic response. In fact, changes in the infrared and Raman spectra are proportional to the mechanical force deforming the force probes, and the relevant bands can be intensified and shifted to a transparent window in the protein spectrum by isotopic substitution. As a result, the proposed molecular force probes can act as "force rulers", allowing the spectroscopic observation and measurement of mechanical forces acting within the proteins under natural conditions without external perturbation. PMID:26928925

  9. Discovery of a Novel Mode of Protein Kinase Inhibition Characterized by the Mesenchymal-epithelial Transition Factor (c-Met) Protein Autophosphorylation by ARQ 197

    SciTech Connect

    S Eathiraj; R Palma; E Volckova; M Hirschi; D France; M Ashwell; T Chan

    2011-12-31

    A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.

  10. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds

    PubMed Central

    Cino, Elio A.; Soares, Iaci N.; Pedrote, Murilo M.; de Oliveira, Guilherme A. P.; Silva, Jerson L.

    2016-01-01

    The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy. PMID:27600721

  11. STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism

    PubMed Central

    Létourneau, Danny; Bédard, Mikaël; Cabana, Jérôme; Lefebvre, Andrée; LeHoux, Jean-Guy; Lavigne, Pierre

    2016-01-01

    START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6. PMID:27340016

  12. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds.

    PubMed

    Cino, Elio A; Soares, Iaci N; Pedrote, Murilo M; de Oliveira, Guilherme A P; Silva, Jerson L

    2016-01-01

    The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy. PMID:27600721

  13. STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism.

    PubMed

    Létourneau, Danny; Bédard, Mikaël; Cabana, Jérôme; Lefebvre, Andrée; LeHoux, Jean-Guy; Lavigne, Pierre

    2016-01-01

    START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through (15)N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6. PMID:27340016

  14. Protein flexibility in the light of structural alphabets.

    PubMed

    Craveur, Pierrick; Joseph, Agnel P; Esque, Jeremy; Narwani, Tarun J; Noël, Floriane; Shinada, Nicolas; Goguet, Matthieu; Leonard, Sylvain; Poulain, Pierre; Bertrand, Olivier; Faure, Guilhem; Rebehmed, Joseph; Ghozlane, Amine; Swapna, Lakshmipuram S; Bhaskara, Ramachandra M; Barnoud, Jonathan; Téletchéa, Stéphane; Jallu, Vincent; Cerny, Jiri; Schneider, Bohdan; Etchebest, Catherine; Srinivasan, Narayanaswamy; Gelly, Jean-Christophe; de Brevern, Alexandre G

    2015-01-01

    Protein structures are valuable tools to understand protein function. Nonetheless, proteins are often considered as rigid macromolecules while their structures exhibit specific flexibility, which is essential to complete their functions. Analyses of protein structures and dynamics are often performed with a simplified three-state description, i.e., the classical secondary structures. More precise and complete description of protein backbone conformation can be obtained using libraries of small protein fragments that are able to approximate every part of protein structures. These libraries, called structural alphabets (SAs), have been widely used in structure analysis field, from definition of ligand binding sites to superimposition of protein structures. SAs are also well suited to analyze the dynamics of protein structures. Here, we review innovative approaches that investigate protein flexibility based on SAs description. Coupled to various sources of experimental data (e.g., B-factor) and computational methodology (e.g., Molecular Dynamic simulation), SAs turn out to be powerful tools to analyze protein dynamics, e.g., to examine allosteric mechanisms in large set of structures in complexes, to identify order/disorder transition. SAs were also shown to be quite efficient to predict protein flexibility from amino-acid sequence. Finally, in this review, we exemplify the interest of SAs for studying flexibility with different cases of proteins implicated in pathologies and diseases. PMID:26075209

  15. Protein flexibility in the light of structural alphabets

    PubMed Central

    Craveur, Pierrick; Joseph, Agnel P.; Esque, Jeremy; Narwani, Tarun J.; Noël, Floriane; Shinada, Nicolas; Goguet, Matthieu; Leonard, Sylvain; Poulain, Pierre; Bertrand, Olivier; Faure, Guilhem; Rebehmed, Joseph; Ghozlane, Amine; Swapna, Lakshmipuram S.; Bhaskara, Ramachandra M.; Barnoud, Jonathan; Téletchéa, Stéphane; Jallu, Vincent; Cerny, Jiri; Schneider, Bohdan; Etchebest, Catherine; Srinivasan, Narayanaswamy; Gelly, Jean-Christophe; de Brevern, Alexandre G.

    2015-01-01

    Protein structures are valuable tools to understand protein function. Nonetheless, proteins are often considered as rigid macromolecules while their structures exhibit specific flexibility, which is essential to complete their functions. Analyses of protein structures and dynamics are often performed with a simplified three-state description, i.e., the classical secondary structures. More precise and complete description of protein backbone conformation can be obtained using libraries of small protein fragments that are able to approximate every part of protein structures. These libraries, called structural alphabets (SAs), have been widely used in structure analysis field, from definition of ligand binding sites to superimposition of protein structures. SAs are also well suited to analyze the dynamics of protein structures. Here, we review innovative approaches that investigate protein flexibility based on SAs description. Coupled to various sources of experimental data (e.g., B-factor) and computational methodology (e.g., Molecular Dynamic simulation), SAs turn out to be powerful tools to analyze protein dynamics, e.g., to examine allosteric mechanisms in large set of structures in complexes, to identify order/disorder transition. SAs were also shown to be quite efficient to predict protein flexibility from amino-acid sequence. Finally, in this review, we exemplify the interest of SAs for studying flexibility with different cases of proteins implicated in pathologies and diseases. PMID:26075209

  16. Chemorheology of phenylboronate-salicylhydroxamate crosslinked hydrogel networks with a sulfonated polymer backbone

    PubMed Central

    Roberts, Meredith C.; Mahalingam, Alamelu; Hanson, Melissa C.; Kiser, Patrick F.

    2012-01-01

    Hydrogel networks crosslinked with polymer-bound phenylboronic acid (PBA) and salicylhydroxamic acid (SHA) demonstrate pH-reversible gel behavior due to the pH-dependent equilibrium of the crosslinking moieties that form the gel network. Furthermore, the pH at which gels behave dynamically can be controlled by use of a polyelectrolyte backbone. Here we report on the frequency-dependent chemorheological characterization of PBA-SHA crosslinked hydrogel networks with a sulfonated polymer backbone. Our results suggest that the anionic nature of the polymers allows reversible crosslinking at neutral pH that an otherwise neutral-backboned PBA-SHA crosslinked network cannot, and that these charge-induced dynamics can be effectively screened by ions in solution. Moreover, moduli-frequency data can effectively be reduced into a single master curve with a neutral-backboned PBA-SHA gel data set as the reference condition. PMID:23132956

  17. Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam.

    PubMed

    Hamuro, Yoshitomo; Molnar, Kathleen S; Coales, Stephen J; OuYang, Bo; Simorellis, Alana K; Pochapsky, Thomas C

    2008-02-01

    Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology. PMID:18023482

  18. Regulation of the epithelial to mesenchymal transition and metastasis by Raf kinase inhibitory protein-dependent Notch1 activity.

    PubMed

    Noh, Hae Sook; Hah, Young-Sool; Ha, Ji Hye; Kang, Min Young; Zada, Sahib; Rha, Sun Young; Kang, Sang Soo; Kim, Hyun Joon; Park, Jae-Yong; Byun, June-Ho; Hahm, Jong Ryeal; Shin, Jeong Kyu; Jeong, Sang-Ho; Lee, Young-Joon; Kim, Deok Ryong

    2016-01-26

    Raf kinase inhibitory protein (RKIP), an endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway, has been implicated as a suppressor of metastasis and a prognostic marker in cancers. However, how RKIP acts as a suppressor during metastasis is not fully understood. Here, we show that RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. The levels of RKIP were significantly decreased in tumor tissues compared to normal tissues, whereas NICD levels were increased. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway. We also found that RKIP directly interacts with Notch1 but has no influence on the intracellular level of the γ-secretase complex that is necessary for Notch1 activation. These data suggest that RKIP plays a distinct role in activation of Notch1 during EMT and metastasis, providing a new target for cancer treatment. PMID:26716415

  19. Regulation of the epithelial to mesenchymal transition and metastasis by Raf kinase inhibitory protein-dependent Notch1 activity

    PubMed Central

    Ha, Ji Hye; Kang, Min Young; Zada, Sahib; Rha, Sun Young; Kang, Sang Soo; Kim, Hyun Joon; Park, Jae-Yong; Byun, June-Ho; Hahm, Jong Ryeal; Shin, Jeong Kyu; Jeong, Sang-Ho; Lee, Young-Joon; Kim, Deok Ryong

    2016-01-01

    Raf kinase inhibitory protein (RKIP), an endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway, has been implicated as a suppressor of metastasis and a prognostic marker in cancers. However, how RKIP acts as a suppressor during metastasis is not fully understood. Here, we show that RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. The levels of RKIP were significantly decreased in tumor tissues compared to normal tissues, whereas NICD levels were increased. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway. We also found that RKIP directly interacts with Notch1 but has no influence on the intracellular level of the γ-secretase complex that is necessary for Notch1 activation. These data suggest that RKIP plays a distinct role in activation of Notch1 during EMT and metastasis, providing a new target for cancer treatment. PMID:26716415

  20. The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition

    PubMed Central

    Tan, E-Jean; Kahata, Kaoru; Idås, Oskar; Thuault, Sylvie; Heldin, Carl-Henrik; Moustakas, Aristidis

    2015-01-01

    The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial–mesenchymal transition (EMT). Transforming growth factor-β (TGFβ) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2′-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion. PMID:25492890

  1. Cold-inducible RNA-binding protein promotes epithelial-mesenchymal transition by activating ERK and p38 pathways.

    PubMed

    Lee, Hae Na; Ahn, Sung-Min; Jang, Ho Hee

    2016-09-01

    Transforming growth factor-β1 (TGF-β1), a potent inducer of epithelial-to-mesenchymal transition (EMT), upregulates the cold-inducible RNA-binding protein (CIRP). The link between CIRP and EMT, however, remains unknown. To determine the role of CIRP in EMT, we performed CIRP knockdown and overexpression experiments in in vitro TGF-β1-induced EMT models. We found that CIRP overexpression promoted the downregulation of epithelial markers and the upregulation of mesenchymal markers after TGF-β1 treatment for EMT induction. It also promoted cell migration and invasion, key features of EMT. In contrast, CIRP knockdown inhibited the downregulation of epithelial markers and the upregulation of mesenchymal markers after TGF-β1 treatment for EMT induction. In addition, it also inhibited cell migration and invasion. Furthermore, we demonstrated that the RNA-recognition motif in CIRP is essential for the role of CIRP in EMT. At the downstream level, CIRP knockdown downregulated Snail, key transcriptional regulator of EMT, while CIRP overexpression upregulated it. We found out that the link between CIRP and Snail is mediated by ERK and p38 pathways. EMT is a critical component of carcinoma metastasis and invasion. As demonstrated in this study, the biological role of CIRP in EMT may explain why CIRP overexpression has been associated with a bad prognosis in cancer patients. PMID:27395339

  2. The Roles of Mitogen-Activated Protein Kinase Pathways in TGF-β-Induced Epithelial-Mesenchymal Transition

    PubMed Central

    Gui, Ting; Sun, Yujing; Shimokado, Aiko; Muragaki, Yasuteru

    2012-01-01

    The mitogen-activated protein kinase (MAPK) pathway allows cells to interpret external signals and respond appropriately, especially during the epithelial-mesenchymal transition (EMT). EMT is an important process during embryonic development, fibrosis, and tumor progression in which epithelial cells acquire mesenchymal, fibroblast-like properties and show reduced intercellular adhesion and increased motility. TGF-β signaling is the first pathway to be described as an inducer of EMT, and its relationship with the Smad family is already well characterized. Studies of four members of the MAPK family in different biological systems have shown that the MAPK and TGF-β signaling pathways interact with each other and have a synergistic effect on the secretion of additional growth factors and cytokines that in turn promote EMT. In this paper, we present background on the regulation and function of MAPKs and their cascades, highlight the mechanisms of MAPK crosstalk with TGF-β signaling, and discuss the roles of MAPKs in EMT. PMID:22363839

  3. Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions

    PubMed Central

    Sunter, Jack D.; Benz, Corinna; Andre, Jane; Whipple, Sarah; McKean, Paul G.; Gull, Keith; Ginger, Michael L.; Lukeš, Julius

    2015-01-01

    ABSTRACT The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure – the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms. PMID:26148511

  4. Gelation of edible blue-green algae protein isolate (Spirulina platensis Strain Pacifica): thermal transitions, rheological properties, and molecular forces involved.

    PubMed

    Chronakis, I S

    2001-02-01

    Proteins isolated from blue-green algae Spirulina platensis strain Pacifica were characterized by visible absorption, differential scanning calorimetry (DSC), viscometry, and dynamic oscillatory rheological measurements. Unique thermal unfolding, denaturation, aggregation, and gelation of the algal protein isolate are presented. DSC analysis showed that thermal transitions occur at about 67 and 109 degrees C at neutral pH. Calcium chloride stabilized the quaternary structure against denaturation and shifted the transitions at higher temperatures. Viscometric studies of Spirulina protein isolate as a function of temperature showed that the onset of the viscosity increase is closely related to the dissociation-denaturation process. Lower viscosities were observed for the protein solutions dissolved at pH 9 due to an increased protein solubility. Solutions of Spirulina protein isolate form elastic gels during heating to 90 degrees C. Subsequent cooling at ambient temperatures caused a further pronounced increase in the elastic moduli and network elasticity. Spirulina protein isolate has good gelling properties with fairly low minimum critical gelling concentrations of about 1.5 and 2.5 wt % in 0.1 M Tris buffer, pH 7, and with 0.02 M CaCl(2) in the same buffer, respectively. It is suggested that mainly the interactions of exposed hydrophobic regions generate the molecular association, initial aggregation, and gelation of the protein isolate during the thermal treatment. Hydrogen bonds reinforce the network rigidity of the protein on cooling and further stabilize the structure of Spirulina protein gels but alone are not sufficient to form a network structure. Intermolecular sulfhydryl and disulfide bonds were found to play a minor role for the network strength of Spirulina protein gels but affect the elasticity of the structures formed. Both time and temperature at isothermal heat-induced gelation within 40-80 degrees C affect substantially the network formation and

  5. Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling

    PubMed Central

    MacDonald, James T.; Kelley, Lawrence A.; Freemont, Paul S.

    2013-01-01

    Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing the degrees of freedom. The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using -carbon positions only and sampling conformations with a Monte Carlo simulated annealing based protocol. Backbone atoms were added using a method previously described and then gradient minimised in the Rosetta force field. Despite the CG potential energy function being sequence-independent, the method performed similarly to methods that explicitly use either fragments of known protein backbones with similar sequences or residue-specific /-maps to restrict the search space. The method was also able to predict with sub-Angstrom accuracy two out of seven loops from recently solved crystal structures of proteins with low sequence and structure similarity to previously deposited structures in the PDB. The ability to sample realistic loop conformations directly from a potential energy function enables the incorporation of additional geometric restraints and the use of more advanced sampling methods in a way that is not possible to do easily with fragment replacement methods and also enable multi-scale simulations for protein design and protein structure prediction. These restraints could be derived from experimental data or could be design restraints in the case of computational protein design. C++ source code is available for download from http

  6. Base sequence dependence and backbone-induced effects on charge transport through DNA

    NASA Astrophysics Data System (ADS)

    Joe, Yong; Lee, Sun; Hedin, Eric

    2009-03-01

    We investigate quantum mechanical electron transmission along the long axis of the DNA molecule using a tight-binding model. Specifically, we use two different DNA models to study the charge transfer efficiency of synthetic ds-DNA. First, the generic form of a simple one-conduction channel model, called the fishbone model, is used. The sugar-phosphate backbone and the coupling amplitude between each site of the base and the backbone are incorporated into an energy-dependent on-site potential in the main DNA site. Here, individual sites represent a base-pair formed by either AT (TA) or GC (CG) pairs coupled via hydrogen bonds. Second, we employ a two-dimensional three-chain model where the backbone on-site energy, the coupling amplitude between the bases and the backbone, and a possible hopping of charge carriers between the successive backbone sites are used as key parameters. The overall transmission and the current-voltage characteristics are calculated to determine the influence of mismatch (impurity) effects in the DNA sequence. Finally, we discuss the transmission gap as a function of coupling between the bases and between the bases and the backbone. *One of the authors (E.R.H) is partially supported by a grant from the Center for Energy Research, Education, and Service (CERES) at Ball State University.

  7. The light-harvesting chlorophyll a/b binding proteins Lhcb1 and Lhcb2 play complementary roles during state transitions in Arabidopsis.

    PubMed

    Pietrzykowska, Malgorzata; Suorsa, Marjaana; Semchonok, Dmitry A; Tikkanen, Mikko; Boekema, Egbert J; Aro, Eva-Mari; Jansson, Stefan

    2014-09-01

    Photosynthetic light harvesting in plants is regulated by phosphorylation-driven state transitions: functional redistributions of the major trimeric light-harvesting complex II (LHCII) to balance the relative excitation of photosystem I and photosystem II. State transitions are driven by reversible LHCII phosphorylation by the STN7 kinase and PPH1/TAP38 phosphatase. LHCII trimers are composed of Lhcb1, Lhcb2, and Lhcb3 proteins in various trimeric configurations. Here, we show that despite their nearly identical amino acid composition, the functional roles of Lhcb1 and Lhcb2 are different but complementary. Arabidopsis thaliana plants lacking only Lhcb2 contain thylakoid protein complexes similar to wild-type plants, where Lhcb2 has been replaced by Lhcb1. However, these do not perform state transitions, so phosphorylation of Lhcb2 seems to be a critical step. In contrast, plants lacking Lhcb1 had a more profound antenna remodeling due to a decrease in the amount of LHCII trimers influencing thylakoid membrane structure and, more indirectly, state transitions. Although state transitions are also found in green algae, the detailed architecture of the extant seed plant light-harvesting antenna can now be dated back to a time after the divergence of the bryophyte and spermatophyte lineages, but before the split of the angiosperm and gymnosperm lineages more than 300 million years ago. PMID:25194026

  8. Uranyl mediated photofootprinting reveals strong E. coli RNA polymerase--DNA backbone contacts in the +10 region of the DeoP1 promoter open complex.

    PubMed Central

    Jeppesen, C; Nielsen, P E

    1989-01-01

    Employing a newly developed uranyl photofootprinting technique (Nielsen et al. (1988) FEBS Lett. 235, 122), we have analyzed the structure of the E. coli RNA polymerase deoP1 promoter open complex. The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region. These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region. The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded. The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region. Images PMID:2503811

  9. Comparison of protein and energy supplementation to mineral supplementation on feeding behavior of grazing cattle during the rainy to the dry season transition.

    PubMed

    Brandão, Rita Kelly Couto; de Carvalho, Gleidson Giordano Pinto; Silva, Robério Rodrigues; Dias, Daniel Lucas Santos; Mendes, Fabrício Bacelar Lima; Lins, Túlio Otávio Jardim D'Almeida; Filho, George Abreu; de Souza, Sinvaldo Oliveira; Barroso, Daniele Soares; de Almeida Rufino, Luana Marta; Tosto, Manuela Silva Libânio

    2016-01-01

    The aim of this study was to evaluate the effects of protein-energy or mineral supplementation on the ingestive behavior of dairy steers on pasture in the post-weaning phase during the rainy to dry season transition. Twenty-two ½ Holstein-Zebu dairy steers with an average initial body weight of 234 ± 16 kg were distributed into a completely randomized design into two groups: protein-energy supplementation and mineral supplementation offered ad libitum. The steers receiving protein-energy supplementation showed higher (P < 0.05) intake of dry matter (DM) and neutral detergent fiber (NDF) than those fed diets composed of mineral salt only. In addition, the animals that received protein-energy supplementation had longer period in grazing and spent on average more time per period eating at the trough (P < 0.05), however no significant differences were observed in the time per period in rumination and time per period in idle (P > 0.05). The supply of protein-energy supplement does not change the feeding behavior, except for an increase in the time spent feeding at the trough. The intake of protein-energy supplement improved the of DM and NDF feed efficiencies in grazing cattle during the rainy to the dry season transition. PMID:27386377

  10. Sequence-specific (1)H, (13)C and (15)N backbone resonance assignments of the plakin repeat domain of human envoplakin.

    PubMed

    Jeeves, Mark; Fogl, Claudia; Al-Jassar, Caezar; Chidgey, Martyn; Overduin, Michael

    2016-04-01

    The plakin repeat domain is a distinctive hallmark of the plakin superfamily of proteins, which are found within all epithelial tissues. Plakin repeat domains mediate the interactions of these proteins with the cell cytoskeleton and are critical for the maintenance of tissue integrity. Despite their biological importance, no solution state resonance assignments are available for any homologue. Here we report the essentially complete (1)H, (13)C and (15)N backbone chemical shift assignments of the singular 22 kDa plakin repeat domain of human envoplakin, providing the means to investigate its interactions with ligands including intermediate filaments. PMID:26590577

  11. Comparison of two adaptive temperature-based replica exchange methods applied to a sharp phase transition of protein unfolding-folding

    NASA Astrophysics Data System (ADS)

    Lee, Michael S.; Olson, Mark A.

    2011-06-01

    Temperature-based replica exchange (T-ReX) enhances sampling of molecular dynamics simulations by autonomously heating and cooling simulation clients via a Metropolis exchange criterion. A pathological case for T-ReX can occur when a change in state (e.g., folding to unfolding of a protein) has a large energetic difference over a short temperature interval leading to insufficient exchanges amongst replica clients near the transition temperature. One solution is to allow the temperature set to dynamically adapt in the temperature space, thereby enriching the population of clients near the transition temperature. In this work, we evaluated two approaches for adapting the temperature set: a method that equalizes exchange rates over all neighbor temperature pairs and a method that attempts to induce clients to visit all temperatures (dubbed "current maximization") by positioning many clients at or near the transition temperature. As a test case, we simulated the 57-residue SH3 domain of alpha-spectrin. Exchange rate equalization yielded the same unfolding-folding transition temperature as fixed-temperature ReX with much smoother convergence of this value. Surprisingly, the current maximization method yielded a significantly lower transition temperature, in close agreement with experimental observation, likely due to more extensive sampling of the transition state.

  12. Solitons and collapse in the λ-repressor protein

    NASA Astrophysics Data System (ADS)

    Krokhotin, Andrey; Lundgren, Martin; Niemi, Antti J.

    2012-08-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding λ-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability, and folding pathways of the λ-repressor protein, which controls the transition from the lysogenic to the lytic state. We first investigate the supersecondary helix-loop helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schrödinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the λ-repressor protein. We introduce a coarse-grained energy function to model the backbone in terms of the Cα atoms and the side chains in terms of the relative orientation of the Cβ atoms. We describe the folding dynamics in terms of relaxation dynamics and find that the folded configuration can be reached from a very generic initial configuration. We conclude that folding is dominated by the temporal ordering of soliton formation. In particular, the third soliton should appear before the first and second. Otherwise, the DNA binding turn does not acquire its correct structure. We confirm the stability of the folded configuration by repeated heating and cooling simulations.

  13. Solitons and collapse in the λ-repressor protein.

    PubMed

    Krokhotin, Andrey; Lundgren, Martin; Niemi, Antti J

    2012-08-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding λ-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability, and folding pathways of the λ-repressor protein, which controls the transition from the lysogenic to the lytic state. We first investigate the supersecondary helix-loop helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schrödinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the λ-repressor protein. We introduce a coarse-grained energy function to model the backbone in terms of the C(α) atoms and the side chains in terms of the relative orientation of the C(β) atoms. We describe the folding dynamics in terms of relaxation dynamics and find that the folded configuration can be reached from a very generic initial configuration. We conclude that folding is dominated by the temporal ordering of soliton formation. In particular, the third soliton should appear before the first and second. Otherwise, the DNA binding turn does not acquire its correct structure. We confirm the stability of the folded configuration by repeated heating and cooling simulations. PMID:23005801

  14. Candida albicans cell shaving uncovers new proteins involved in cell wall integrity, yeast to hypha transition, stress response and host-pathogen interaction

    PubMed Central

    Hernáez, María Luisa; Reales-Calderon, Jose Antonio; Solis, Norma V.; Filler, Scott G.; Monteoliva, Lucia; Gil, Concha

    2015-01-01

    The ability to switch from yeast to hyphal growth is essential for virulence in Candida albicans. The cell surface is the initial point of contact between the fungus and the host. In this work, a free-gel proteomic strategy based on tryptic digestion of live yeast and hyphae cells and protein identification using LC-MS/MS methodology was used to identify cell surface proteins. Using this strategy, a total of 943 proteins were identified, of which 438 were in yeast and 928 were in hyphae. Of these proteins, 79 were closely related to the organization and biogenesis of the cell wall, including 28 GPI-anchored proteins, such as Hyr1 and Sod5 which were detected exclusively in hyphae, and Als2 and Sap10which were detected only in yeast. A group of 17 proteins of unknown function were subsequently studied by analysis of the corresponding deletion mutants. We found that four new proteins, Pst3, Tos1, Orf19.3060 and Orf19.5352 are involved in cell wall integrity and in C. albicans’ engulfment by macrophages. Moreover, the putative NADH-ubiquinone-related proteins, Ali1, Mci4, Orf19.287 and Orf19.7590, are also involved in osmotic and oxidative resistance, yeast to hypha transition and the ability to damage and invade oral epithelial cells. PMID:26087349

  15. Prognostic significance of epithelial-mesenchymal transition proteins Twist and Foxc2 in phyllodes tumours of the breast.

    PubMed

    Lim, Jeffrey Chun Tatt; Koh, Valerie Cui Yun; Tan, Jane Sie Yong; Tan, Wai Jin; Thike, Aye Aye; Tan, Puay Hoon

    2015-02-01

    Epithelial-mesenchymal transition (EMT), an important process during embryonic development, is reportedly exploited during tumour progression. Deregulation of EMT-related molecules has been shown in many malignancies, including breast carcinoma. We aim to investigate the clinical relevance and prognostic significance of EMT proteins, Twist and Foxc2, in breast phyllodes tumours (PTs). The study cohort comprised 271 PTs diagnosed from 2003 to 2010. Of these, 188 (69.4 %) were benign, 60 (22.1 %) borderline, and 23 (8.5 %) malignant. Immunohistochemistry for Twist and Foxc2 was performed on tissue microarray sections. Percentage of tumour cells stained was evaluated and correlated with clinicopathological parameters and clinical outcome. Twist and Foxc2 stromal nuclear expression was associated with tumour grade (P = 0.038 and 0.012). Foxc2 stromal nuclear expression was positively correlated with epithelial expression (P < 0.001), tumour relapse, and metastasis (P = 0.037). Furthermore, stromal nuclear immunoreactivity of Twist and Foxc2 was interrelated (P < 0.001). Tumours expressing Foxc2 and those co-expressing both Twist and Foxc2 revealed a shorter time to recurrence (P < 0.001 and 0.001) and death (P = 0.044 and 0.015). Twist and Foxc2 stromal expression in PTs was significantly correlated with tumour grade and worse histological features. In addition, expression of Foxc2 and co-expression of Twist and Foxc2 in the stroma of PTs contributed to poorer prognosis. Clinical relevance of EMT-related molecules may be worthy of further investigation in PTs. PMID:25677742

  16. The Zurich Tradition: Backbone of the Wolf Number Series (Invited)

    NASA Astrophysics Data System (ADS)

    Friedli, T. K.

    2013-12-01

    of the Wolf archive we examined and corrected the calibrations of the original observations by Wolf during the Bernese and early Zurich period to the countings by Wolf with the handheld Parisian refractor and the transition from those observations to Wolfer and the transition from Wolfer to Brunner. For the observations of the late Wolf some drift in the counting practice probably due to a degradation of acuity is obvious. A correction is not easy. Nor is a thorough examination of the transition from Brunner to Waldmeier, since the original countings of Waldmeier are still missing.

  17. Design and Synthesis of Peptide YY Analogues with C-terminal Backbone Amide-to-Ester Modifications

    PubMed Central

    2013-01-01

    Peptide YY (PYY) is a gut hormone that activates the G protein-coupled neuropeptide Y (NPY) receptors, and because of its appetite reducing actions, it is evaluated as an antiobesity drug candidate. The C-terminal tail of PYY is crucial for activation of the NPY receptors. Here, we describe the design and preparation of a series of PYY(3–36) depsipeptide analogues, in which backbone amide-to-ester modifications were systematically introduced in the C-terminal. Functional NPY receptor assays and circular dichroism revealed that the ψ(CONH) bonds at positions 30–31 and 33–34 are particularly important for receptor interaction and that the latter is implicated in Y2 receptor selectivity. PMID:24900634

  18. Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

    PubMed

    Sharma, Rakhi; Sahu, Bhubanananda; Ray, Malay K; Deshmukh, Mandar V

    2015-04-01

    Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W. PMID:24496608

  19. (1)H, (13)C and (15)N backbone assignment of the EC-1 domain of human E-cadherin.

    PubMed

    Prasasty, Vivitri D; Krause, Mary E; Tambunan, Usman S F; Anbanandam, Asokan; Laurence, Jennifer S; Siahaan, Teruna J

    2015-04-01

    The Extracellular 1 (EC1) domain of E-cadherin has been shown to be important for cadherin-cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell-cell adhesion located at the adherens junction of the biological barriers (i.e., intestinal mucosa and the blood-brain barrier (BBB)). Cadherin peptides can modulate cadherin interactions to improve drug delivery through the BBB. However, the mechanism of modulating the E-cadherin interactions by cadherin peptides has not been fully elucidated. To provide a basis for subsequent examination of the structure and peptide-binding properties of the EC1 domain of human E-cadherin using solution NMR spectroscopy, the (1)H, (13)C and (15)N backbone resonance of the uniformly labeled-EC1 were assigned and the secondary structure was determined based on the chemical shift values. These resonance assignments are essential for assessing protein-ligand interactions and are reported here. PMID:24510398

  20. Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations.

    PubMed

    Rosta, Edina; Nowotny, Marcin; Yang, Wei; Hummer, Gerhard

    2011-06-15

    We use quantum mechanics/molecular mechanics simulations to study the cleavage of the ribonucleic acid (RNA) backbone catalyzed by ribonuclease H. This protein is a prototypical member of a large family of enzymes that use two-metal catalysis to process nucleic acids. By combining Hamiltonian replica exchange with a finite-temperature string method, we calculate the free energy surface underlying the RNA-cleavage reaction and characterize its mechanism. We find that the reaction proceeds in two steps. In a first step, catalyzed primarily by magnesium ion A and its ligands, a water molecule attacks the scissile phosphate. Consistent with thiol-substitution experiments, a water proton is transferred to the downstream phosphate group. The transient phosphorane formed as a result of this nucleophilic attack decays by breaking the bond between the phosphate and the ribose oxygen. In the resulting intermediate, the dissociated but unprotonated leaving group forms an alkoxide coordinated to magnesium ion B. In a second step, the reaction is completed by protonation of the leaving group, with a neutral Asp132 as a likely proton donor. The overall reaction barrier of ∼15 kcal mol(-1), encountered in the first step, together with the cost of protonating Asp132, is consistent with the slow measured rate of ∼1-100/min. The two-step mechanism is also consistent with the bell-shaped pH dependence of the reaction rate. The nonmonotonic relative motion of the magnesium ions along the reaction pathway agrees with X-ray crystal structures. Proton-transfer reactions and changes in the metal ion coordination emerge as central factors in the RNA-cleavage reaction. PMID:21539371

  1. Catalytic Mechanism of RNA Backbone Cleavage by Ribonuclease H from QM/MM Simulations

    PubMed Central

    Rosta, Edina; Nowotny, Marcin; Yang, Wei; Hummer, Gerhard

    2011-01-01

    We use quantum mechanics/molecular mechanics (QM/MM) simulations to study the cleavage of the ribonucleic acid (RNA) backbone catalyzed by ribonuclease H. This protein is a prototypical member of a large family of enzymes that use two-metal catalysis to process nucleic acids. By combining Hamiltonian replica exchange with a finite-temperature string method, we calculate the free energy surface underlying the RNA cleavage reaction and characterize its mechanism. We find that the reaction proceeds in two steps. In a first step, catalyzed primarily by magnesium ion A and its ligands, a water molecule attacks the scissile phosphate. Consistent with thiol-substitution experiments, a water proton is transferred to the downstream phosphate group. The transient phosphorane formed as a result of this nucleophilic attack decays by breaking the bond between the phosphate and the ribose oxygen. In the resulting intermediate, the dissociated but unprotonated leaving group forms an alkoxide coordinated to magnesium ion B. In a second step, the reaction is completed by protonation of the leaving group, with a neutral Asp132 as a likely proton donor. The overall reaction barrier of ~15 kcal mol−1, encountered in the first step, together with the cost of protonating Asp132, is consistent with the slow measured rate of ~1–100/min. The two-step mechanism is also consistent with the bell-shaped pH dependence of the reaction rate. The non-monotonic relative motion of the magnesium ions along the reaction pathway agrees with X-ray crystal structures. Proton transfer reactions and changes in the metal ion coordination emerge as central factors in the RNA cleavage reaction. PMID:21539371

  2. Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases.

    PubMed

    Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Zeuner, Birgitte; Łężyk, Mateusz; Difilippo, Elisabetta; Logtenberg, Madelon J; Schols, Henk A; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

    2015-10-01

    This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The β-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest homologues are 59 % for HEX1 and 51 % for HEX2 on the protein level. Both β-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-β-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-β-N-acetylgalactosamine (pNP-GalNAc) and exhibit pH optima of 8 and 6 for HEX1 and HEX2, respectively. The enzymes are able to hydrolyze N-acetylchitooligosaccharides with a degree of polymerization of two, three, and four. The major findings were, that HEX1 and HEX2 catalyze trans-glycosylation reactions with lactose as acceptor, giving rise to the human milk oligosaccharide precursor lacto-N-triose II (LNT2) with yields of 2 and 8 % based on the donor substrate. In total, trans-glycosylation reactions were tested with the disaccharide acceptors β-lactose, sucrose, and maltose, as well as with the monosaccharides galactose and glucose resulting in the successful attachment of GlcNAc to the acceptor in all cases. PMID:25843303

  3. Solution structure and backbone dynamics of streptopain: insight into diverse substrate specificity.

    PubMed

    Wang, Chih-Chieh; Houng, Hsiang-Chee; Chen, Chun-Liang; Wang, Pei-Ju; Kuo, Chih-Feng; Lin, Yee-Shin; Wu, Jiunn-Jong; Lin, Ming T; Liu, Ching-Chuan; Huang, Wenya; Chuang, Woei-Jer

    2009-04-17

    Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease expressed by Streptococcus pyogenes. The D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed to study the effect of the allelic variants on their protease activity. In contrast to other mutants, the G239D mutant was approximately 12-fold less active. The Gly-239 residue is located within the C-terminal S230-G239 region, which cannot be observed in the x-ray structure. The three-dimensional structure and backbone dynamics of the 28-kDa mature SPE B (mSPE B) were determined. Unlike the x-ray structure of the 40-kDa zymogen SPE B (proSPE B), we observed the interactions between the C-terminal loop and the active site residues in mSPE B. The structural differences between mSPE B and proSPE B were the conformation of the C-terminal loop and the orientation of the catalytic His-195 residue, suggesting that activation and inactivation of SPE B is involved in the His-195 side-chain rotation. Dynamics analysis of mSPE B and the mSPE B/inhibitor complexes showed that the catalytic and C-terminal loops were the most flexible regions with low order parameter values of 0.5 to 0.8 and exhibited the motion on the ps/ns timescale. These findings suggest that the flexible C-terminal loop of SPE B may play an important role in controlling the substrate binding, resulting in its broad substrate specificity. PMID:19237546

  4. Plants Actively Avoid State Transitions upon Changes in Light Intensity: Role of Light-Harvesting Complex II Protein Dephosphorylation in High Light1[OPEN

    PubMed Central

    Suorsa, Marjaana; Rantala, Marjaana; Aro, Eva-Mari

    2015-01-01

    Photosystem II (PSII) core and light-harvesting complex II (LHCII) proteins in plant chloroplasts undergo reversible phosphorylation upon changes in light intensity (being under control of redox-regulated STN7 and STN8 kinases and TAP38/PPH1 and PSII core phosphatases). Shift of plants from growth light to high light results in an increase of PSII core phosphorylation, whereas LHCII phosphorylation concomitantly decreases. Exactly the opposite takes place when plants are shifted to lower light intensity. Despite distinct changes occurring in thylakoid protein phosphorylation upon light intensity changes, the excitation balance between PSII and photosystem I remains unchanged. This differs drastically from the canonical-state transition model induced by artificial states 1 and 2 lights that concomitantly either dephosphorylate or phosphorylate, respectively, both the PSII core and LHCII phosphoproteins. Analysis of the kinase and phosphatase mutants revealed that TAP38/PPH1 phosphatase is crucial in preventing state transition upon increase in light intensity. Indeed, tap38/pph1 mutant revealed strong concomitant phosphorylation of both the PSII core and LHCII proteins upon transfer to high light, thus resembling the wild type under state 2 light. Coordinated function of thylakoid protein kinases and phosphatases is shown to secure balanced excitation energy for both photosystems by preventing state transitions upon changes in light intensity. Moreover, PROTON GRADIENT REGULATION5 (PGR5) is required for proper regulation of thylakoid protein kinases and phosphatases, and the pgr5 mutant mimics phenotypes of tap38/pph1. This shows that there is a close cooperation between the redox- and proton gradient-dependent regulatory mechanisms for proper function of the photosynthetic machinery. PMID:25902812

  5. Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

    PubMed

    Zhang, Shi Qun; Zou, Zui; Shen, Hui; Shen, Shuai Shuai; Miao, Qi; Huang, Xin; Liu, Wei; Li, Li Ping; Chen, Si Min; Yan, Lan; Zhang, Jun Dong; Zhao, Jing Jun; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-05-01

    The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3)-glucan, a crucial pathogen-associated molecular pattern (PAMP) of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans. PMID:27144456

  6. Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity

    PubMed Central

    Zhang, Shi Qun; Zou, Zui; Shen, Hui; Shen, Shuai Shuai; Miao, Qi; Huang, Xin; Liu, Wei; Li, Li Ping; Chen, Si Min; Yan, Lan; Zhang, Jun Dong; Zhao, Jing Jun; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3)-glucan, a crucial pathogen-associated molecular pattern (PAMP) of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans. PMID:27144456

  7. Diffraction in resonant electron scattering from helical macromolecules: Effects of the DNA backbone

    SciTech Connect

    Caron, Laurent; Sanche, Leon

    2005-09-15

    We recently developed a theoretical framework to treat low-energy electron scattering from helical macromolecules. In this article, we use this framework to extend our previous model of simple base-pair scatterers, organized into the DNA structure, to include the backbone. The internal diffraction pattern due to base pairs is still present, but addition of the backbone screens the base pairs by a factor of 2. More interestingly, the effect of constructive interference on the phosphate groups within the backbone itself is seen to be strong at lower energies. We perform a calculation for electrons incident perpendicular and parallel to the axis of a fragment and find comparable electron patterns on the phosphate groups at the surface of films consisting of vertically or horizontally arranged segments relative to the substrate.

  8. Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

    PubMed Central

    Feagin, Trevor A.; Shah, Nirmal I.; Heemstra, Jennifer M.

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  9. Convenient and scalable synthesis of fmoc-protected Peptide nucleic Acid backbone.

    PubMed

    Feagin, Trevor A; Shah, Nirmal I; Heemstra, Jennifer M

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  10. Polyboramines for Hydrogen Release: Polymers Containing Lewis Pairs in their Backbone.

    PubMed

    Ledoux, Audrey; Larini, Paolo; Boisson, Christophe; Monteil, Vincent; Raynaud, Jean; Lacôte, Emmanuel

    2015-12-21

    The one-step polycondensation of diamines and diboranes triggered by the in situ deprotonation of the diammonium salts and concomitant reduction of bisboronic acids leads to the assembly of polymer chains through multiple Lewis pairing in their backbone. These new polyboramines are dihydrogen reservoirs that can be used for the hydrogenation of imines and carbonyl compounds. They also display a unique dihydrogen thermal release profile that is a direct consequence of the insertion of the amine-borane linkages in the polymeric backbone. PMID:26563914

  11. Electric field induced localization phenomena in a ladder network with superlattice configuration: Effect of backbone environment

    NASA Astrophysics Data System (ADS)

    Dutta, Paramita; Maiti, Santanu K.; Karmakar, S. N.

    2014-09-01

    Electric field induced localization properties of a tight-binding ladder network in presence of backbone sites are investigated. Based on Green's function formalism we numerically calculate two-terminal transport together with density of states for different arrangements of atomic sites in the ladder and its backbone. Our results lead to a possibility of getting multiple mobility edges which essentially plays a switching action between a completely opaque to fully or partly conducting region upon the variation of system Fermi energy, and thus, support in fabricating mesoscopic or DNA-based switching devices.

  12. Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

    SciTech Connect

    Wu, B.-K.; Li, C.-C.; Chen, H.-J.; Chang, J.-L.; Jeng, K.-S.; Chou, C.-K.; Hsu, M.-T.; Tsai, T.-F. . E-mail: tftsai@ym.edu.tw

    2006-02-17

    The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and {alpha}-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides First physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice.

  13. Effect of supplementation of beef cattle with different protein levels and degradation rates during transition from the dry to rainy season.

    PubMed

    Fernandes, Rodolfo Maciel; de Almeida, Chafic Mustafé; Carvalho, Bruna Caldas; Alves Neto, João Alexandrino; Mota, Verônica Aparecida Costa; de Resende, Flávio Dutra; Siqueira, Gustavo Rezende

    2016-01-01

    The objective of this study was to evaluate the effect of increasing the supply of protein with different degradation rates on the performance and metabolism of growing Nellore cattle reared on Brachiaria brizantha cv. Marandu pasture during the transition period from the dry to rainy season. The experiment was installed on an area of 34 ha, divided into 12 paddocks with an average area of 2.85 ha. In the performance evaluation were utilized 72 recently weaned, non-castrated Nellore cattle with an initial body weight (BW) of 199 kg (SEM = 16). The following supplements were used: energy protein supplement containing 25% crude protein (CP) (C-25) and energy protein supplements containing 40% CP with one third highly degradable CP and two thirds poorly degradable CP (40-1/3NPN), one half highly degradable CP and one half poorly degradable CP (40-1/2NPN), and two thirds highly degradable CP and one third poorly degradable CP (40-2/3NPN). Higher protein degradation rates reduced supplement intake (P < 0.01). In the first period, animals consuming supplement 40-1/3NPN exhibited higher average daily gain (ADG) (0.30 kg/day), similar to that of animals receiving supplement 40-1/2NPN (P = 0.04). In the second period, supplement 40-2/3NPN resulted in lower ADG (0.19 kg/day less than the other supplements). There was no effect of supplement on animal performance in the third period (P > 0.10), when ADG was 0.56 kg/day. In conclusion, the response to supplementation is associated with interactions with characteristics of the forage canopy. Supplementation with a true protein source will be beneficial only during the early stage of the dry-rainy season transition period. PMID:26433723

  14. A Novel RNA-Recognition-Motif Protein Is Required for Premeiotic G1/S-Phase Transition in Rice (Oryza sativa L.)

    PubMed Central

    Nonomura, Ken-Ichi; Eiguchi, Mitsugu; Nakano, Mutsuko; Takashima, Kazuya; Komeda, Norio; Fukuchi, Satoshi; Miyazaki, Saori; Miyao, Akio; Hirochika, Hirohiko; Kurata, Nori

    2011-01-01

    The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species. PMID:21253568

  15. Design of HIV Protease Inhibitors Targeting Protein Backbone: An Effective Strategy for Combating Drug Resistance

    SciTech Connect

    Ghosh, Arun K.; Chapsal, Bruno D.; Weber, Irene T.; Mitsuya, Hiroaki

    2008-06-03

    The discovery of human immunodeficiency virus (HIV) protease inhibitors (PIs) and their utilization in highly active antiretroviral therapy (HAART) have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

  16. Mapping of Post-translational Modifications of Transition Proteins, TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2*

    PubMed Central

    Gupta, Nikhil; Madapura, M. Pradeepa; Bhat, U. Anayat; Rao, M. R. Satyanarayana

    2015-01-01

    In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg71, Arg75, and Arg92 residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys88 and Lys91 residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome. PMID:25818198

  17. Mapping of Post-translational Modifications of Transition Proteins, TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2.

    PubMed

    Gupta, Nikhil; Madapura, M Pradeepa; Bhat, U Anayat; Rao, M R Satyanarayana

    2015-05-01

    In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg(71), Arg(75), and Arg(92) residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys(88) and Lys(91) residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome. PMID:25818198

  18. Influence of the polymer backbone structure on the properties of aromatic ionomers with pendant sulfobenzoyl side chains for use as proton-exchange membranes.

    PubMed

    Jutemar, Elin Persson; Jannasch, Patric

    2010-12-01

    Six different ionomers having various aromatic polymer backbones with pendant 2-sulfobenzoyl side chains were prepared by nucleophilic aromatic substitution reactions of lithium 2,6-difluoro-2'-sulfobenzophenone with 4,4-biphenol, 2,7-dihydroxynaphthalene, 4,4-isopropylidenediphenol, 4,4-dihydroxydiphenyl ether, 4,4'-thiodiphenol, and 4,4'-thiobisbenzenethiol, respectively, to produce four poly(arylene ether)s, one poly(arylene ether sulfide), and one poly(arylene sulfide). Mechanically tough proton-exchange membranes with ion-exchange capacities in the narrow range from 1.9 to 2.3 mequiv/g were cast from the high-molecular-weight ionomers, and subsequently investigated with respect to their structure-property relationships. Glass transitions were only detected for ionomers in the sodium salt form, and increasing glass-transition temperatures (Tg) were found to give higher thermal decomposition temperatures. Analysis by small-angle X-ray scattering indicated that the ionic clustering was promoted for ionomers with flexible polymer backbones and low Tg values. The proton conductivity of the membranes at 80 °C under fully humidified conditions was found between 0.02 and 0.2 S/cm and appeared to depend primarily on the Tg. PMID:21138250

  19. Lasso peptide, a highly stable structure and designable multifunctional backbone.

    PubMed

    Zhao, Ning; Pan, Yongxu; Cheng, Zhen; Liu, Hongguang

    2016-06-01

    Lasso peptide belongs to a new class of natural product with highly compact and stable structure. It has varieties of biological activities, among which the most important one is its antibacterial efficacy. Novel lasso peptides have been constantly discovered and analyzed by advanced techniques, and the biosynthesis or even chemical synthesis of lasso peptide has been studied after learning its constituent amino acids and maturation process. Structural identification of lasso peptide provides information for elucidating the mechanisms of its antibacterial activity and basis for further modifications. Ring of lasso peptide is the key to both its highly compact and stable structure and its intrinsic antibacterial property. The loop has been considered as suitable modification region of lasso peptide, such as V11-S18 of MccJ25 being modifiable without disrupting the lasso structure in biosynthesis. The tail is the immunity protein that can export lasso peptide out of its produced strain and serve as a self-protection mechanism at the same time. Most of currently known lasso peptides are non-pathogenic, which implies that the modified lasso peptides are promising candidates for medical applications. Arginine, glycine, and aspartic acid as a ligands of cancer-specific receptor have been grafted to the loop of lasso peptide without losing its bioactivity, and many other targets are expected to be used for lasso peptide modification. Multi-molecular modification and large-scale production need to be studied and solved in future for designing and using multifunctional lasso peptide based on its extraordinary stable structure. PMID:27074719

  20. Function of Amphiphilic Biomolecular Machines: Elastic Protein-based Polymers

    NASA Astrophysics Data System (ADS)

    Urry, Dan W.

    2000-03-01

    Elastic protein-based polymers function as biomolecular machines due to inverse temperature transitions of hydrophobic folding and assembly. The transitions occur either on raising the temperature from below to above the transition temperature, Tt, or on isothermally lowering Tt from above to below an operating temperature. The inverse temperature transition involves a decrease in entropy of the polymer component of the system on raising the temperature and a larger increase in solvent entropy on hydrophobic association. Tt depends on the quantity of hydrophobic hydration that undergoes transition to bulk water. Designed amphiphilic polymers perform free energy transductions involving the intensive variables of mechanical force, pressure, temperature, chemical potential, electrochemical potential and electromagnetic radiation and define a set of five axioms for their function as machines. The physical basis for these diverse energy conversions is competition for hydration between apolar (hydrophobic) and polar (e.g., charged) moieties. The effectiveness of these Tt-type entropic elastic protein-based machines is due to repeating peptide sequences that form regular, dynamic repeating structures and exhibit damping of backbone torsional oscillations on extension.

  1. Phase Transitions in the Nucleus: the functional implications of concentration-dependent assembly of a Liquid-like RNA/Protein Body

    NASA Astrophysics Data System (ADS)

    Zhu, Lian; Weber, Stephanie; Berry, Joel; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford

    2015-03-01

    The nucleolus is a liquid-like membrane-less nuclear body which plays an important role in cell growth and size control. By modulating nucleolar component concentration through RNAi conditions that change C. elegans cell size, we find that nucleoli only assemble above a threshold concentration; moreover, the ripening dynamics of nucleated droplets are consistent with the hypothesis that the assembly of the nucleolus represents an intracellular liquid-liquid phase transition. A key question is how this phase-transition is linked to the primary function of the nucleolus, in transcribing and processing ribosomal RNA. To address this, we characterize the localization of RNA Polymerase I, a key transcriptional enzyme, into nucleolar foci as a function of nucleolar component concentration. Our results suggest that there are a small number of key disordered phosphoproteins that may serve as a link between transcription and assembly. Finally, we present preliminary results using a reduced model system consisting of purified nucleolar proteins to assess the ability of nucleolar proteins to drive liquid-liquid phase separation in vitro. These results lay the foundation for a quantitative understanding of intracellular phase transitions and their impact on biomedically-critical RNA-processing steps.

  2. Structural Mechanism Underlying the Specific Recognition between the Arabidopsis State-Transition Phosphatase TAP38/PPH1 and Phosphorylated Light-Harvesting Complex Protein Lhcb1[OPEN

    PubMed Central

    Wei, Xuepeng; Guo, Jiangtao; Li, Mei; Liu, Zhenfeng

    2015-01-01

    During state transitions, plants regulate energy distribution between photosystems I and II through reversible phosphorylation and lateral migration of the major light-harvesting complex LHCII. Dephosphorylation of LHCII and the transition from state 2 to state 1 requires a thylakoid membrane-associated phosphatase named TAP38 or PPH1. TAP38/PPH1 specifically targets LHCII but not the core subunits of photosystem II, whereas the underlying molecular mechanism of their mutual recognition is currently unclear. Here, we present the structures of Arabidopsis thaliana TAP38/PPH1 in the substrate-free and substrate-bound states. The protein contains a type 2C serine/threonine protein phosphatase (PP2C) core domain, a Mn2+ (or Mg2+) binuclear center and two additional motifs contributing to substrate recognition. A 15-mer phosphorylated N-terminal peptide of Lhcb1 binds to TAP38/PPH1 on two surface clefts enclosed by the additional motifs. The first segment of the phosphopeptide is clamped by a pair of tooth-like arginine residues at Cleft 1 site. The binding adopts the lock-and-key mechanism with slight rearrangement of the substrate binding residues on TAP38/PPH1. Meanwhile, a more evident substrate-induced fitting occurs on Cleft 2 harboring the extended part of the phosphopeptide. The results unravel the bases for the specific recognition between TAP38/PPH1 and phosphorylated Lhcb1, a crucial step in state transitions. PMID:25888588

  3. Spatial and temporal resolution of interfaces, phase transitions and isolation of three families of proteins in calcium carbonate based biocomposite materials

    NASA Astrophysics Data System (ADS)

    Belcher, Angela M.

    The abalone shell is a microlaminate organic-inorganic composite of mineral and biopolymers exhibiting exceptional nanoscale regularity and a strength ˜3,000 times greater than that of the inorganic crystals themselves. Although the integral proteins typically comprise less than 2% (by mass) of the shell, they determine the structural organization and properties of the mineralized composite. Three families of proteins from the red abalone shell have been isolated, purified and characterized and their role in the formation of this composite structure have been studied. I have found that Nature uses two different mechanisms for directing structure during biofabrication of the shell and flat pearl. One controls structure and orientation of the mineral at the atomic and nanoscale levels; and the other controls ordering over macroscale dimensions. The proteins of these three families exert this control: (1) a calcite crystal-nucleating protein (nuclein) (2) polyanionic proteins that determine the phase, orientation, and morphology of calcite and aragonite; and (3) matrix envelope proteins that determine the lamellar spacing and crystal size of aragonite in nacre. Preliminary sequence analysis of these proteins and their cloned genes reveals several unusual structural features. Use of the purified nucleating and polyanionic proteins allow us, in vitro, to abruptly and sequentially switch crystallographic phase from calcite to aragonite and vice-versa, producing multiphase composites with micron-scale domains. Development of in vivo (flat pearl) and in vitro systems allowed for the elucidation of organic-inorganic interfaces and phase transitions of the growing shell to better understand the biomineralization process and development of new multiphase composites.

  4. Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

    NASA Astrophysics Data System (ADS)

    Fujisawa, Tetsuro; Ueda, Toshifumi; Kameyama, Keiichi; Aso, Yoichi; Ishiguro, Ryo

    2013-06-01

    The small heat shock proteins (sHSPs) solubilize thermo-denatured proteins without adenosine triphosphate energy consumption to facilitate protein refolding. sHSP20.8 is one of the silkworm (Bombyx mori) sHSPs having only one cystein in the N-terminal domain: Cys43. We report a simple measurement of oligomeric transition of sHSP20.8 using high hydrostatic pressure native polyacrylamide gel electrophoresis (high hydrostatic pressure (HP) native polyacrylamide gel electrophoresis (PAGE)). At ambient pressure under oxydative condition, the native PAGE of thermal transition of sHSP20.8 oligomer displayed a cooperative association. In contrast, HP native PAGE clearly demonstrated that sHSP20.8 dissociated at 80 MPa and 25°C, and the resultant molecular species gradually reassociated with time under that condition. In addition, the reassociation process was suppressed in the presence of the reductant. These results are consistent with the idea that sHSP20.8 oligomer temporally dissociates at the first thermo-sensing step and reassociates with the oxidation of Cys43.

  5. CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

    SciTech Connect

    Miura, Yuka; Hagiwara, Natsumi; Radisky, Derek C.; Hirai, Yohei

    2014-09-10

    Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.

  6. Synthesis and evaluation of backbone/amide-modified analogs of leualacin.

    PubMed

    Hu, M K; Yang, F C; Chou, C C; Yen, M H

    1999-02-22

    Leualacin (1), a cyclic depsi-pentapeptide, and its backbone/amide-modified analogs 2-4 were synthesized. Amide analogue 3 exhibited stronger vasodilatory effects. It also strongly inhibited collagen- and arachidonic acid (AA)-induced platelet aggregations with IC50s of 0.6 microM and 2.0 microM, respectively. PMID:10098664

  7. Animals without Backbones: The Invertebrate Story. Grade Level 5-9.

    ERIC Educational Resources Information Center

    Jerome, Brian; Fuqua, Paul

    This guide, when used in tandem with the videotape "Animals Without Backbones," helps students learn about invertebrates. These materials promote hands-on discovery and learning. The guide is composed of six curriculum-based teaching units: (1) "Getting Started"; (2) "Porifera"; (3) "Cnidarians"; (4) "Worms"; (5) "Mollusks"; (6) "Arthropods"; and…

  8. Graduate Education in Kinesiology: Are We Part of "America's Backbone for Competitiveness and Innovation"?

    ERIC Educational Resources Information Center

    DePauw, Karen P.

    2008-01-01

    Graduate education in the United States has been identified as being the backbone of American competitiveness and innovation in a recent report by the Council of Graduate Schools. The report provides a framework for examining the role of graduate education in partnership with business and government to advance an action agenda for achieving…

  9. Data supporting beta-amyloid dimer structural transitions and protein-lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures.

    PubMed

    Cheng, Sara Y; Chou, George; Buie, Creighton; Vaughn, Mark W; Compton, Campbell; Cheng, Kwan H

    2016-06-01

    This data article supports the research article entitled "Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface" [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes. PMID:27054174

  10. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  11. Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes.

    PubMed

    Popova, Antoaneta V; Hundertmark, Michaela; Seckler, Robert; Hincha, Dirk K

    2011-07-01

    Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates. PMID:21443857

  12. Advanced oxidation protein products induce endothelial-to-mesenchymal transition in human renal glomerular endothelial cells through induction of endoplasmic reticulum stress.

    PubMed

    Liang, Xiujie; Duan, Na; Wang, Yue; Shu, Shuangshuang; Xiang, Xiaohong; Guo, Tingting; Yang, Lei; Zhang, Shaojie; Tang, Xun; Zhang, Jun

    2016-01-01

    Endothelial-to-mesenchymal transition (EndMT) in renal glomerular endothelial cells plays a critical role in the pathogenesis of diabetic nephropathy (DN). Furthermore, advanced oxidation protein products (AOPPs) have been shown to contribute to the progression of DN. However, whether AOPPs induce EndMT in renal glomerular endothelial cells remains unclear. Thus, we investigated the effect of AOPPs on human renal glomerular endothelial cells (HRGECs) and the mechanisms underlying the effects. Our results showed that AOPP treatment lowered the expression of vascular endothelial cadherin, CD31, and claudin 5 and induced the overexpression of α-smooth muscle actin, vimentin, and fibroblast-specific protein 1, which indicated that AOPPs induced EndMT in HRGECs. Furthermore, AOPP stimulation increased the expression of glucose-regulated protein 78 and CCAAT/enhancer-binding protein-homologous protein, which suggested that AOPPs triggered endoplasmic reticulum (ER) stress in HRGECs. Notably, the aforementioned AOPP effects were reversed following the treatment of cells with salubrinal, an inhibitor of ER stress, whereas the effects were reproduced after exposure to thapsigargin, an inducer of ER stress. Collectively, our results indicate that AOPPs trigger EndMT in HRGECs through the induction of ER stress. These findings suggest novel therapeutic strategies for inhibiting renal fibrosis by targeting ER stress. PMID:26861949

  13. A commercial human protamine-2 antibody used in several studies to detect mouse protamine-2 recognizes mouse transition protein-2 but not protamine-2.

    PubMed

    Eckhardt, Matthias; Wang-Eckhardt, Lihua

    2015-11-01

    The exchange of histones for transition proteins (TNPs) and finally protamines is an essential process during spermatogenesis that enables the strong condensation of chromatin during sperm formation. Research on this process obviously depends on the availability of specific antibodies recognizing these nuclear proteins. A commercial antibody generated against human protamine-2 (PRM2) has been described to cross-react with mouse PRM2 and in fact has been used in several studies to detect mouse PRM2. Some inconsistent results obtained with this goat-derived antibody prompted us to re-examine its specificity. In immunofluorescence experiments with epididymal sperm, only a low percentage of sperm nuclei were stained by this antibody, whereas a mouse monoclonal anti- PRM2 antibody stained most sperm, as expected. Western blot analysis of basic nuclear proteins from spermatids and sperm separated by acid urea (AU) gel electrophoresis revealed that the goat anti- PRM2 antiserum binds to mouse TNP2 but not mouse PRM2. Epitope mapping using glutathione-S-transferase-fusion proteins with peptide sequences conserved in human PRM2 and mouse TNP2 identified the tetrapeptide arginyl-lysyl-arginyl-threonine as an epitope of the goat anti- PRM2 antiserum. Our findings underline the importance of using AU gel electrophoresis to confirm specificities of antibodies directed against basic nuclear proteins, which are not well separated, and may show abnormal migration behaviour, in SDS-polyacrylamide gel electrophoresis. PMID:26268249

  14. Molecular dynamics studies of protein folding and aggregation

    NASA Astrophysics Data System (ADS)

    Ding, Feng

    that globular proteins under a denaturing environment partially unfold and aggregate by forming stabilizing hydrogen bonds between the backbones of the partial folded substructures. Proteins or peptides rich in alpha-helices also aggregate into beta-rich amyloid fibrils. Upon aggregation, the protein or peptide undergoes a conformational transition from alpha-helices to beta-sheets. The transition of alpha-helix to beta-hairpin (two-stranded beta-sheet) is studied in an all-heavy-atom discrete molecular dynamics model of a polyalanine chain. An entropical driving scenario for the alpha-helix to beta-hairpin transition is discovered.

  15. The Construction of Metal-Organic Framework with Active Backbones by the Utilization of Reticular Chemistry

    NASA Astrophysics Data System (ADS)

    Choi, Eunwoo

    With the principles of reticular chemistry, metal-organic frameworks with ultra-high porosity, chiral-recognition unit as a chiral stationary phase, metalloporhyrins for enhanced hydrogen adsorption and an intrinsic conductivity to form porous conductors, have been prepared. This dissertation presents how the principles of reticular chemistry were utilized to achieve in the preparations of metal-organic frameworks with a large surface area and active backbones. Through the simple isoreticular (having the same framework topology) expansion from MOF-177 composed with 1,3,5-tris(4'-carboxyphenyl-)benzene (BTB3-) as the strut; MOF-200 was prepared with 4,4',4"-(benzene-1,3,5-triyl-tris(benzene-4,1-diy1))tribenzoic acid an extension from BTB3- by a phenylene unit to yield one of the most porous MOFs with a Langmuir surface area of 10,400 m2. and the lowest density of 0.22 cm3.g-1. A successful thermal polymerization reaction at 325 °C inside of the pores of highly porous MOF, MOF-177, was performed and verified the integrity of the MOF structure even after the thermal reaction. 1,4-Diphenylbutadiyne that is known to polymerize upon heating to form a conjugated backbone was impregnated via solution-diffusion into MOF-177 and then subsequently polymerized by heat to form polymer impregnated MOF-177. Characterization was carried out using powder X-ray diffraction and volumetric sorption analyzer. MOF-1020 with a linear quaterphenyl dicarboxylate-based strut was designed to contain a chiral bisbinaphthyl crown-ether moiety for alkyl ammonium resolution was precisely placed into a Zn4O(CO2)6-based cubic MOF structure. Unfortunately, the chiral resolution was not achieved due to the sensitivity and the pore environment of MOF-1020. However, an interesting phenomenon was observed, where the loss of crystallinity occurs upon solvent removal while the crystallites remain shiny and crystalline, but it readily is restored upon re-solvation of the crystallites. This rare

  16. Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells.

    PubMed

    Palma, Camila de Souza; Grassi, Mariana Lopes; Thomé, Carolina Hassibe; Ferreira, Germano Aguiar; Albuquerque, Daniele; Pinto, Mariana Tomazini; Ferreira Melo, Fernanda Ursoli; Kashima, Simone; Covas, Dimas Tadeu; Pitteri, Sharon J; Faça, Vitor M

    2016-03-01

    Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. He