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Sample records for protein backbone transitions

  1. Nonlinear backbone torsional pair correlations in proteins

    PubMed Central

    Long, Shiyang; Tian, Pu

    2016-01-01

    Protein allostery requires dynamical structural correlations. Physical origin of which, however, remain elusive despite intensive studies during last two and half decades. Based on analysis of molecular dynamics (MD) simulation trajectories for ten proteins with different sizes and folds, we found that nonlinear backbone torsional pair (BTP) correlations, which are mainly spatially long-ranged and are dominantly executed by loop residues, exist extensively in most analyzed proteins. Examination of torsional motion for correlated BTPs suggested that such nonlinear correlations are mainly associated aharmonic torsional state transitions and in some cases strongly anisotropic local torsional motion of participating torsions, and occur on widely different and relatively longer time scales. In contrast, correlations between backbone torsions in stable α helices and β strands are mainly linear and spatially short-ranged, and are more likely to associate with harmonic local torsional motion. Further analysis revealed that the direct cause of nonlinear contributions are heterogeneous linear correlations. These findings implicate a general search strategy for novel allosteric modulation sites of protein activities. PMID:27708342

  2. Nonlinear backbone torsional pair correlations in proteins

    NASA Astrophysics Data System (ADS)

    Long, Shiyang; Tian, Pu

    2016-10-01

    Protein allostery requires dynamical structural correlations. Physical origin of which, however, remain elusive despite intensive studies during last two and half decades. Based on analysis of molecular dynamics (MD) simulation trajectories for ten proteins with different sizes and folds, we found that nonlinear backbone torsional pair (BTP) correlations, which are mainly spatially long-ranged and are dominantly executed by loop residues, exist extensively in most analyzed proteins. Examination of torsional motion for correlated BTPs suggested that such nonlinear correlations are mainly associated aharmonic torsional state transitions and in some cases strongly anisotropic local torsional motion of participating torsions, and occur on widely different and relatively longer time scales. In contrast, correlations between backbone torsions in stable α helices and β strands are mainly linear and spatially short-ranged, and are more likely to associate with harmonic local torsional motion. Further analysis revealed that the direct cause of nonlinear contributions are heterogeneous linear correlations. These findings implicate a general search strategy for novel allosteric modulation sites of protein activities.

  3. 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

  4. Structural dependencies of protein backbone 2JNC' couplings.

    PubMed

    Juranić, Nenad; Dannenberg, J J; Cornilescu, Gabriel; Salvador, Pedro; Atanasova, Elena; Ahn, Hee-Chul; Macura, Slobodan; Markley, John L; Prendergast, Franklyn G

    2008-04-01

    Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated (2)J(NC') couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H-bond strengths derived from (h3)J(NC') couplings, provides useful insight into the overall energy profile of the protein backbone in solution.

  5. 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

  6. 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

  7. 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

  8. Relaxation of backbone bond geometry improves protein energy landscape modeling.

    PubMed

    Conway, Patrick; Tyka, Michael D; DiMaio, Frank; Konerding, David E; Baker, David

    2014-01-01

    A key issue in macromolecular structure modeling is the granularity of the molecular representation. A fine-grained representation can approximate the actual structure more accurately, but may require many more degrees of freedom than a coarse-grained representation and hence make conformational search more challenging. We investigate this tradeoff between the accuracy and the size of protein conformational search space for two frequently used representations: one with fixed bond angles and lengths and one that has full flexibility. We performed large-scale explorations of the energy landscapes of 82 protein domains under each model, and find that the introduction of bond angle flexibility significantly increases the average energy gap between native and non-native structures. We also find that incorporating bonded geometry flexibility improves low resolution X-ray crystallographic refinement. These results suggest that backbone bond angle relaxation makes an important contribution to native structure energetics, that current energy functions are sufficiently accurate to capture the energetic gain associated with subtle deformations from chain ideality, and more speculatively, that backbone geometry distortions occur late in protein folding to optimize packing in the native state.

  9. 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}).

  10. Retrieving backbone string neighbors provides insights into structural modeling of membrane proteins.

    PubMed

    Sun, Jiang-Ming; Li, Tong-Hua; Cong, Pei-Sheng; Tang, Sheng-Nan; Xiong, Wen-Wei

    2012-07-01

    Identification of protein structural neighbors to a query is fundamental in structure and function prediction. Here we present BS-align, a systematic method to retrieve backbone string neighbors from primary sequences as templates for protein modeling. The backbone conformation of a protein is represented by the backbone string, as defined in Ramachandran space. The backbone string of a query can be accurately predicted by two innovative technologies: a knowledge-driven sequence alignment and encoding of a backbone string element profile. Then, the predicted backbone string is employed to align against a backbone string database and retrieve a set of backbone string neighbors. The backbone string neighbors were shown to be close to native structures of query proteins. BS-align was successfully employed to predict models of 10 membrane proteins with lengths ranging between 229 and 595 residues, and whose high-resolution structural determinations were difficult to elucidate both by experiment and prediction. The obtained TM-scores and root mean square deviations of the models confirmed that the models based on the backbone string neighbors retrieved by the BS-align were very close to the native membrane structures although the query and the neighbor shared a very low sequence identity. The backbone string system represents a new road for the prediction of protein structure from sequence, and suggests that the similarity of the backbone string would be more informative than describing a protein as belonging to a fold.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Modeling 15N NMR chemical shift changes in protein backbone with pressure

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

  18. 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

  19. Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

    PubMed

    Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

    2016-02-01

    Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation.

  20. 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

  1. 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.

  2. Pseudo 5D HN(C)N experiment to facilitate the assignment of backbone resonances in proteins exhibiting high backbone shift degeneracy

    NASA Astrophysics Data System (ADS)

    Kumar, Dinesh; Raikwal, Nisha; Shukla, Vaibhav Kumar; Pandey, Himanshu; Arora, Ashish; Guleria, Anupam

    2014-09-01

    Assignment of protein backbone resonances is most routinely carried out using triple resonance three-dimensional NMR experiments involving amide 1H/15N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high-degree of backbone shift degeneracy. In this backdrop, a novel reduced-dimensionality (RD) experiment -(5, 3)D-hNCO-CANH- is presented to facilitate/validate the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide 15N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. HiNi and Hi-1Ni-1) for overlapping amide-NH peaks. The experiment -in combination with routine triple resonance 3D-NMR experiments involving backbone amide (1H/15N) and carbon (13Cα/13C‧) chemical shifts- will serve as a powerful complementary tool to achieve the nearly complete assignment of protein backbone resonances in a time efficient manner.

  3. 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

  4. 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.

  5. Effects of Protein Stabilizing Agents on Thermal Backbone Motions: A Disulfide Trapping Study†

    PubMed Central

    Butler, Scott L.; Falke, Joseph J.

    2010-01-01

    Chemical stabilizers are widely used to enhance protein stability, both in nature and in the laboratory. Here, the molecular mechanism of chemical stabilizers is studied using a disulfide trapping assay to measure the effects of stabilizers on thermal backbone dynamics in the Escherichia coli galactose/glucose binding protein. Two types of backbone fluctuations are examined: (a) relative movements of adjacent surface α-helices within the same domain and (b) interdomain twisting motions. Both types of fluctuations are significantly reduced by all six stabilizers tested (glycerol, sucrose, trehalose, l-glucose, d-glucose, and d-galactose), and in each case larger amplitude motions are inhibited more than smaller ones. Motional inhibition does not require a high-affinity stabilizer binding site, indicating that the effects of stabilizers are nonspecific. Overall, the results support the theory that effective stabilizing agents act by favoring the most compact structure of a protein, thereby reducing local backbone fluctuations away from the fully folded state. Such inhibition of protein backbone dynamics may be a general mechanism of protein stabilization in extreme thermal or chemical environments. PMID:8718847

  6. Protein backbone torsion angle-based structure comparison and secondary structure database web server.

    PubMed

    Jung, Sunghoon; Bae, Se-Eun; Ahn, Insung; Son, Hyeon S

    2013-09-01

    Structural information has been a major concern for biological and pharmaceutical studies for its intimate relationship to the function of a protein. Three-dimensional representation of the positions of protein atoms is utilized among many structural information repositories that have been published. The reliability of the torsional system, which represents the native processes of structural change in the structural analysis, was partially proven with previous structural alignment studies. Here, a web server providing structural information and analysis based on the backbone torsional representation of a protein structure is newly introduced. The web server offers functions of secondary structure database search, secondary structure calculation, and pair-wise protein structure comparison, based on a backbone torsion angle representation system. Application of the implementation in pair-wise structural alignment showed highly accurate results. The information derived from this web server might be further utilized in the field of ab initio protein structure modeling or protein homology-related analyses.

  7. A unified NMR strategy for high-throughput determination of backbone fold of small proteins.

    PubMed

    Kumar, Dinesh; Gautam, Anmol; Hosur, Ramakrishna V

    2012-12-01

    An efficient semi-automated strategy called PFBD (i.e. Protein Fold from Backbone Data only) has been presented for rapid backbone fold determination of small proteins. It makes use of NMR parameters involving backbone atoms only. These include chemical shifts, amide-amide NOEs and H-bonds. The backbone chemical shifts are obtained in an automated manner from the orthogonal 2D projections of variants of HNN and HN(C)N experiments (Kumar et al., in Magn Reson Chem 50(5):357-363, 2012) using AUTOBA (Borkar et al. in J Biomol NMR 50(3):285-297, 2011); backbone H-bonds are manually derived from constant time long-range 2D-HnCO spectrum (Cordier and Grzesiek in J Am Chem Soc 121:1601-1602, 1999); and amide-amide NOEs are derived from 3D HNCO NOESY experiment which provides NOEs along the direct (1)H dimension that has maximum resolution (Lohr and Ruterjans in J Biomol NMR 9(1):371-388, 1997). All the experiments needed for the execution of PFBD can be recorded and analyzed in about 24-48 h depending upon the concentration of the protein and dispersion of amide cross-peaks in the (1)H-(15)N correlation spectrum. Thus, we believe that the strategy, because of its speed and simplicity will be very valuable in Biomolecular NMR community for high-throughput structural proteomics of small folded proteins of MW < 10-12 kDa, the regime where NMR is generally preferred over X-ray crystallography. The strategy has been validated and demonstrated here on two small globular proteins: human ubiquitin (76 aa) and chicken SH3 domain (62 aa). PMID:23054485

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

    PubMed

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

    2015-05-01

    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 (O(2) NH ) 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 (O(2) NH  < 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, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH 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 O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements.

  9. 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

  10. RASP: rapid and robust backbone chemical shift assignments from protein structure.

    PubMed

    MacRaild, Christopher A; Norton, Raymond S

    2014-03-01

    Chemical shift prediction has an unappreciated power to guide backbone resonance assignment in cases where protein structure is known. Here we describe Resonance Assignment by chemical Shift Prediction (RASP), a method that exploits this power to derive protein backbone resonance assignments from chemical shift predictions. Robust assignments can be obtained from a minimal set of only the most sensitive triple-resonance experiments, even for spectroscopically challenging proteins. Over a test set of 154 proteins RASP assigns 88 % of residues with an accuracy of 99.7 %, using only information available from HNCO and HNCA spectra. Applied to experimental data from a challenging 34 kDa protein, RASP assigns 90 % of manually assigned residues using only 40 % of the experimental data required for the manual assignment. RASP has the potential to significantly accelerate the backbone assignment process for a wide range of proteins for which structural information is available, including those for which conventional assignment strategies are not feasible. PMID:24445369

  11. Protein backbone angle restraints from searching a database for chemical shift and sequence homology.

    PubMed

    Cornilescu, G; Delaglio, F; Bax, A

    1999-03-01

    Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C alpha, 13C beta, 13C', 1H alpha and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar phi and psi backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 degrees. Approximately 3% of the predictions made by TALOS are found to be in error.

  12. 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

  13. 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.

  14. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks.

    PubMed

    Shen, Yang; Bax, Ad

    2013-07-01

    A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, ≥90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed (ϕ, ψ) torsion angles of ca 12º. TALOS-N also reports sidechain χ(1) rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.

  15. Repacking protein cores with backbone freedom: structure prediction for coiled coils.

    PubMed

    Harbury, P B; Tidor, B; Kim, P S

    1995-08-29

    Progress in homology modeling and protein design has generated considerable interest in methods for predicting side-chain packing in the hydrophobic cores of proteins. Present techniques are not practically useful, however, because they are unable to model protein main-chain flexibility. Parameterization of backbone motions may represent a general and efficient method to incorporate backbone relaxation into such fixed main-chain models. To test this notion, we introduce a method for treating explicitly the backbone motions of alpha-helical bundles based on an algebraic parameterization proposed by Francis Crick in 1953 [Crick, F. H. C. (1953) Acta Crystallogr. 6, 685-689]. Given only the core amino acid sequence, a simple calculation can rapidly reproduce the crystallographic main-chain and core side-chain structures of three coiled coils (one dimer, one trimer, and one tetramer) to within 0.6-A root-mean-square deviations. The speed of the predictive method [approximately 3 min per rotamer choice on a Silicon Graphics (Mountain View, CA) 4D/35 computer] permits it to be used as a design tool.

  16. Effect of Nanoconfinement on the Glass Transition Temperature and Small Molecule Diffusion in Polymers of Varying Backbone Stiffness

    NASA Astrophysics Data System (ADS)

    Deng, Hui; Mundra, Manish; Torkelson, John

    2010-03-01

    Fluorescence spectroscopy was used to determine the glass transition temperature in ultrathin supported bisphenol-A polysulfone (BPAPS) and bisphenol-A polycarbonate (BPAPC) films and compared to previous results for ultrathin supported polystyrene (PS) films. BPAPC and BPAPS are more rigid than PS due to the presence of aromatic rings in their polymer backbones. A dramatic increase in Tg-reduction upon confinement was seen for polymers with increased backbone stiffness. A fluorescence-multilayer film technique was then used to determine the diffusion coefficient of a small molecule probe in ultrathin supported PS films. A decrease in the diffusion coefficient of the probe was observed upon confinement of the PS films. This procedure is also being applied to ultrathin supported BPAPC and BPAPS films to explore the impact of polymer backbone rigidity on small molecule diffusion in nanoconfined polymers.

  17. RosettaBackrub—a web server for flexible backbone protein structure modeling and design

    PubMed Central

    Lauck, Florian; Smith, Colin A.; Friedland, Gregory F.; Humphris, Elisabeth L.; Kortemme, Tanja

    2010-01-01

    The RosettaBackrub server (http://kortemmelab.ucsf.edu/backrub) implements the Backrub method, derived from observations of alternative conformations in high-resolution protein crystal structures, for flexible backbone protein modeling. Backrub modeling is applied to three related applications using the Rosetta program for structure prediction and design: (I) modeling of structures of point mutations, (II) generating protein conformational ensembles and designing sequences consistent with these conformations and (III) predicting tolerated sequences at protein–protein interfaces. The three protocols have been validated on experimental data. Starting from a user-provided single input protein structure in PDB format, the server generates near-native conformational ensembles. The predicted conformations and sequences can be used for different applications, such as to guide mutagenesis experiments, for ensemble-docking approaches or to generate sequence libraries for protein design. PMID:20462859

  18. Computer assignment of the backbone resonances of labelled proteins using two-dimensional correlation experiments.

    PubMed

    Morelle, N; Brutscher, B; Simorre, J P; Marion, D

    1995-02-01

    We present ALPS (Assignment for Labelled Protein Spectra), a flexible computer program for the automatic assignment of backbone NMR resonances of (15)N/(13)C-labelled proteins. The program constructs pseudoresidues from peak-picking lists of a set of two-dimensional triple resonance experiments and uses either a systematic search or a simulated annealing-based optimization to perform the assignment. This method has been successfully tested on two-dimensional triple resonance spectra of Rhodobacter capsulatus ferrocytochrome c (2) (116 amino acids).

  19. Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

    PubMed

    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.

  20. 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

  1. A practical implementation of cross-spectrum in protein backbone resonance assignment.

    PubMed

    Chen, Kang; Delaglio, Frank; Tjandra, Nico

    2010-04-01

    The concept of cross-spectrum is applied in protein NMR spectroscopy to assist in the backbone sequential resonance assignment. Cross-spectrum analysis is used routinely to reveal correlations in frequency domains as a means to reveal common features contained in multiple time series. Here the cross-spectrum between related NMR spectra, for example HNCO and HN(CA)CO, can be calculated with point-by-point multiplications along their common C' carbon axis. In the resulting higher order cross-spectrum, an enhanced correlation signal occurs at every common i-1 carbon frequency allowing the amide proton H(N) (and nitrogen N) resonances from residues i and i-1 to be identified. The cross-spectrum approach is demonstrated using 2D spectra H(N)CO, H(NCA)CO, H(NCO)CACB, and H(N)CACB measured on a 15N/13C double-labeled Ubiquitin sample. These 2D spectra are used to calculate two pseudo-3D cross-spectra, H(i)-H(i)(-1)-C'(i)(-1) and H(i)-H(i)(-1)-CA(i)(-1)CB(i)(-1). We show using this approach, backbone resonances of H, C', CA, and CB can be fully assigned without ambiguity. The cross-spectrum principle is expected to offer an easy, practical, and more quantitative approach for heteronuclear backbone resonance assignment. PMID:20053573

  2. Structure and assembly of group B streptococcus pilus 2b backbone protein.

    PubMed

    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.

  3. 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

  4. BEST-HNN and 2D-(HN) NH experiments for rapid backbone assignment in proteins

    NASA Astrophysics Data System (ADS)

    Kumar, Dinesh; Paul, Subhradip; Hosur, Ramakrishna V.

    2010-05-01

    HNN has proven to be an extremely valuable experiment for rapid and unambiguous backbone (H N, 15N) assignment in ( 13C, 15N) labeled proteins. However, low sensitivity of the experiment is often a limiting factor, especially when the transverse relaxation times ( T2) are short. We show here that BEST modification Schanda et al. (2006) [2] increases the sensitivity per unit time by more than a factor of 2.0 and thus substantially increases the speed of data collection; good 3D data can be collected in 8-10 h. Next, we present a simple method for amino-acid type identification based on simple 2D versions of the HNN experiment, labeled here as 2D-(HN) NH. Each of these experiments which produce anchor points for Gly, Ala, Ser/Thr residues, can be recorded in less than an hour. These enable rapid data acquisition, rapid analysis, and consequently rapid assignment of backbone (H N, 15N) resonances. The 2D-(HN) NH experiment does not involve aliphatic/aromatic protons and hence can be applied to deuterated protein samples as well, which is an additional advantage. The experiments have been demonstrated with human ubiquitin (76 aa) and acetic-acid denatured HIV-1 protease (99 aa), as representatives of folded and unfolded protein systems, respectively.

  5. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts.

    PubMed

    Shen, Yang; Delaglio, Frank; Cornilescu, Gabriel; Bax, Ad

    2009-08-01

    NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between 13C, 15N and 1H chemical shifts and backbone torsion angles phi and psi (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted phi and psi angles, equals +/-13 degrees . Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

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

    DOE PAGES

    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. 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.

  8. Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments.

    PubMed

    Wenrich, Broc R; Sonstrom, Reilly E; Gupta, Riju A; Rovnyak, David

    2015-11-01

    Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%(w/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.

  9. "Chameleonic" backbone hydrogen bonds in protein binding and as drug targets.

    PubMed

    Menéndez, C A; Accordino, S R; Gerbino, D C; Appignanesi, G A

    2015-10-01

    We carry out a time-averaged contact matrix study to reveal the existence of protein backbone hydrogen bonds (BHBs) whose net persistence in time differs markedly form their corresponding PDB-reported state. We term such interactions as "chameleonic" BHBs, CBHBs, precisely to account for their tendency to change the structural prescription of the PDB for the opposite bonding propensity in solution. We also find a significant enrichment of protein binding sites in CBHBs, relate them to local water exposure and analyze their behavior as ligand/drug targets. Thus, the dynamic analysis of hydrogen bond propensity might lay the foundations for new tools of interest in protein binding-site prediction and in lead optimization for drug design. PMID:26486885

  10. Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction.

    PubMed

    Amsden, Jason J; Kralj, Joel M; Chieffo, Logan R; Wang, Xihua; Erramilli, Shyamsunder; Spudich, Elena N; Spudich, John L; Ziegler, Lawrence D; Rothschild, Kenneth J

    2007-10-11

    Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport. PMID:17880126

  11. Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity

    PubMed Central

    Ollikainen, Noah; de Jong, René M.; Kortemme, Tanja

    2015-01-01

    Interactions between small molecules and proteins play critical roles in regulating and facilitating diverse biological functions, yet our ability to accurately re-engineer the specificity of these interactions using computational approaches has been limited. One main difficulty, in addition to inaccuracies in energy functions, is the exquisite sensitivity of protein–ligand interactions to subtle conformational changes, coupled with the computational problem of sampling the large conformational search space of degrees of freedom of ligands, amino acid side chains, and the protein backbone. Here, we describe two benchmarks for evaluating the accuracy of computational approaches for re-engineering protein-ligand interactions: (i) prediction of enzyme specificity altering mutations and (ii) prediction of sequence tolerance in ligand binding sites. After finding that current state-of-the-art “fixed backbone” design methods perform poorly on these tests, we develop a new “coupled moves” design method in the program Rosetta that couples changes to protein sequence with alterations in both protein side-chain and protein backbone conformations, and allows for changes in ligand rigid-body and torsion degrees of freedom. We show significantly increased accuracy in both predicting ligand specificity altering mutations and binding site sequences. These methodological improvements should be useful for many applications of protein – ligand design. The approach also provides insights into the role of subtle conformational adjustments that enable functional changes not only in engineering applications but also in natural protein evolution. PMID:26397464

  12. Flexible backbone sampling methods to model and design protein alternative conformations.

    PubMed

    Ollikainen, Noah; Smith, Colin A; Fraser, James S; Kortemme, Tanja

    2013-01-01

    Sampling alternative conformations is key to understanding how proteins work and engineering them for new functions. However, accurately characterizing and modeling protein conformational ensembles remain experimentally and computationally challenging. These challenges must be met before protein conformational heterogeneity can be exploited in protein engineering and design. Here, as a stepping stone, we describe methods to detect alternative conformations in proteins and strategies to model these near-native conformational changes based on backrub-type Monte Carlo moves in Rosetta. We illustrate how Rosetta simulations that apply backrub moves improve modeling of point mutant side-chain conformations, native side-chain conformational heterogeneity, functional conformational changes, tolerated sequence space, protein interaction specificity, and amino acid covariation across protein-protein interfaces. We include relevant Rosetta command lines and RosettaScripts to encourage the application of these types of simulations to other systems. Our work highlights that critical scoring and sampling improvements will be necessary to approximate conformational landscapes. Challenges for the future development of these methods include modeling conformational changes that propagate away from designed mutation sites and modulating backbone flexibility to predictively design functionally important conformational heterogeneity.

  13. Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis.

    PubMed

    Mack, J W; Usha, M G; Long, J; Griffin, R G; Wittebort, R J

    2000-01-01

    We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.

  14. Using halogen bonds to address the protein backbone: a systematic evaluation.

    PubMed

    Wilcken, Rainer; Zimmermann, Markus O; Lange, Andreas; Zahn, Stefan; Boeckler, Frank M

    2012-08-01

    Halogen bonds are specific embodiments of the sigma hole bonding paradigm. They represent directional interactions between the halogens chlorine, bromine, or iodine and an electron donor as binding partner. Using quantum chemical calculations at the MP2 level, we systematically explore how they can be used in molecular design to address the omnipresent carbonyls of the protein backbone. We characterize energetics and directionality and elucidate their spatial variability in sub-optimal geometries that are expected to occur in protein-ligand complexes featuring a multitude of concomitant interactions. By deriving simple rules, we aid medicinal chemists and chemical biologists in easily exploiting them for scaffold decoration and design. Our work shows that carbonyl-halogen bonds may be used to expand the patentable medicinal chemistry space, redefining halogens as key features. Furthermore, this data will be useful for implementing halogen bonds into pharmacophore models or scoring functions making the QM information available for automatic molecular recognition in virtual high throughput screening.

  15. Optimization of Protein Backbone Dihedral Angles by Means of Hamiltonian Reweighting

    PubMed Central

    2016-01-01

    Molecular dynamics simulations depend critically on the accuracy of the underlying force fields in properly representing biomolecules. Hence, it is crucial to validate the force-field parameter sets in this respect. In the context of the GROMOS force field, this is usually achieved by comparing simulation data to experimental observables for small molecules. In this study, we develop new amino acid backbone dihedral angle potential energy parameters based on the widely used 54A7 parameter set by matching to experimental J values and secondary structure propensity scales. In order to find the most appropriate backbone parameters, close to 100 000 different combinations of parameters have been screened. However, since the sheer number of combinations considered prohibits actual molecular dynamics simulations for each of them, we instead predicted the values for every combination using Hamiltonian reweighting. While the original 54A7 parameter set fails to reproduce the experimental data, we are able to provide parameters that match significantly better. However, to ensure applicability in the context of larger peptides and full proteins, further studies have to be undertaken. PMID:27559757

  16. 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.

  17. 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.

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

    PubMed

    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 C(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.

  19. OLIGOMERIZATION OF A RETROVIRAL MATRIX PROTEIN IS FACILITATED BY BACKBONE FLEXIBILITY ON NS TIMESCALE

    PubMed Central

    Srb, Pavel; Vlach, Jiří; Prchal, Jan; Grocký, Marián; Ruml, Tomáš; Lang, Jan; Hrabal, Richard

    2011-01-01

    Oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidences have been obtained that the oligomerization capacity of the wild type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on ns timescale. Increased flexibility has been observed for two regions: the loop between α-helices α2 and α3 and the C-terminal half of α-helix α3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural pre-organization play an important role in the control of the oligomerization process. PMID:21366213

  20. Solution structure and backbone dynamics of the defunct domain of calcium vector protein.

    PubMed

    Théret, I; Baladi, S; Cox, J A; Gallay, J; Sakamoto, H; Craescu, C T

    2001-11-20

    CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes. PMID:11705378

  1. 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

  2. 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.

  3. ANGLOR: A Composite Machine-Learning Algorithm for Protein Backbone Torsion Angle Prediction

    PubMed Central

    Wu, Sitao; Zhang, Yang

    2008-01-01

    We developed a composite machine-learning based algorithm, called ANGLOR, to predict real-value protein backbone torsion angles from amino acid sequences. The input features of ANGLOR include sequence profiles, predicted secondary structure and solvent accessibility. In a large-scale benchmarking test, the mean absolute error (MAE) of the phi/psi prediction is 28°/46°, which is ∼10% lower than that generated by software in literature. The prediction is statistically different from a random predictor (or a purely secondary-structure-based predictor) with p-value <1.0×10−300 (or <1.0×10−148) by Wilcoxon signed rank test. For some residues (ILE, LEU, PRO and VAL) and especially the residues in helix and buried regions, the MAE of phi angles is much smaller (10–20°) than that in other environments. Thus, although the average accuracy of the ANGLOR prediction is still low, the portion of the accurately predicted dihedral angles may be useful in assisting protein fold recognition and ab initio 3D structure modeling. PMID:18923703

  4. Role of evolutionary information in prediction of aromatic-backbone NH interactions in proteins.

    PubMed

    Kaur, Harpreet; Raghava, G P S

    2004-04-23

    In this study, an attempt has been made to develop a neural network-based method for predicting segments in proteins containing aromatic-backbone NH (Ar-NH) interactions using multiple sequence alignment. We have analyzed 3121 segments seven residues long containing Ar-NH interactions, extracted from 2298 non-redundant protein structures where no two proteins have more than 25% sequence identity. Two consecutive feed-forward neural networks with a single hidden layer have been trained with standard back-propagation as learning algorithm. The performance of the method improves from 0.12 to 0.15 in terms of Matthews correlation coefficient (MCC) value when evolutionary information (multiple alignment obtained from PSI-BLAST) is used as input instead of a single sequence. The performance of the method further improves from MCC 0.15 to 0.20 when secondary structure information predicted by PSIPRED is incorporated in the prediction. The final network yields an overall prediction accuracy of 70.1% and an MCC of 0.20 when tested by five-fold cross-validation. Overall the performance is 15.2% higher than the random prediction. The method consists of two neural networks: (i) a sequence-to-structure network which predicts the aromatic residues involved in Ar-NH interaction from multiple alignment of protein sequences and (ii) a structure-to structure network where the input consists of the output obtained from the first network and predicted secondary structure. Further, the actual position of the donor residue within the 'potential' predicted fragment has been predicted using a separate sequence-to-structure neural network. Based on the present study, a server Ar_NHPred has been developed which predicts Ar-NH interaction in a given amino acid sequence. The web server Ar_NHPred is available at and (mirror site).

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

    PubMed

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

    2015-07-03

    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.

  6. 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

  7. 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

  8. 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.

  9. The determinants of bond angle variability in protein/peptide backbones: A comprehensive statistical/quantum mechanics analysis.

    PubMed

    Improta, Roberto; Vitagliano, Luigi; Esposito, Luciana

    2015-11-01

    The elucidation of the mutual influence between peptide bond geometry and local conformation has important implications for protein structure refinement, validation, and prediction. To gain insights into the structural determinants and the energetic contributions associated with protein/peptide backbone plasticity, we here report an extensive analysis of the variability of the peptide bond angles by combining statistical analyses of protein structures and quantum mechanics calculations on small model peptide systems. Our analyses demonstrate that all the backbone bond angles strongly depend on the peptide conformation and unveil the existence of regular trends as function of ψ and/or φ. The excellent agreement of the quantum mechanics calculations with the statistical surveys of protein structures validates the computational scheme here employed and demonstrates that the valence geometry of protein/peptide backbone is primarily dictated by local interactions. Notably, for the first time we show that the position of the H(α) hydrogen atom, which is an important parameter in NMR structural studies, is also dependent on the local conformation. Most of the trends observed may be satisfactorily explained by invoking steric repulsive interactions; in some specific cases the valence bond variability is also influenced by hydrogen-bond like interactions. Moreover, we can provide a reliable estimate of the energies involved in the interplay between geometry and conformations.

  10. Predicting protein backbone chemical shifts from Cα coordinates: extracting high resolution experimental observables from low resolution models.

    PubMed

    Frank, Aaron T; Law, Sean M; Ahlstrom, Logan S; Brooks, Charles L

    2015-01-13

    Given the demonstrated utility of coarse-grained modeling and simulations approaches in studying protein structure and dynamics, developing methods that allow experimental observables to be directly recovered from coarse-grained models is of great importance. In this work, we develop one such method that enables protein backbone chemical shifts (1HN, 1Hα, 13Cα, 13C, 13Cβ, and 15N) to be predicted from Cα coordinates. We show that our Cα-based method, LARMORCα, predicts backbone chemical shifts with comparable accuracy to some all-atom approaches. More importantly, we demonstrate that LARMORCα predicted chemical shifts are able to resolve native structure from decoy pools that contain both native and non-native models, and so it is sensitive to protein structure. As an application, we use LARMORCα to characterize the transient state of the fast-folding protein gpW using recently published NMR relaxation dispersion derived backbone chemical shifts. The model we obtain is consistent with the previously proposed model based on independent analysis of the chemical shift dispersion pattern of the transient state. We anticipate that LARMORCα will find utility as a tool that enables important protein conformational substates to be identified by “parsing” trajectories and ensembles generated using coarse-grained modeling and simulations.

  11. Observation of persistent α-helical content and discrete types of backbone disorder during a molten globule to ordered peptide transition via deep-UV resonance Raman spectroscopy

    PubMed Central

    Brown, Mia C.; Mutter, Andrew; Koder, Ronald L.; JiJi, Renee D.; Cooley, Jason W.

    2016-01-01

    The molten globule state can aide in the folding of a protein to a functional structure and is loosely defined as an increase in structural disorder with conservation of the ensemble secondary structure content. Simultaneous observation of persistent secondary structure content with increased disorder has remained experimentally problematic. As a consequence, modeling how the molten globule state remains stable and how it facilitates proper folding remains difficult due to a lack of amenable spectroscopic techniques to characterize this class of partially unfolded proteins. Previously, deep-UV resonance Raman (dUVRR) spectroscopy has proven useful in the resolution of global and local structural fluctuations in the secondary structure of proteins. In this work, dUVRR was employed to study the molten globule to ordered transition of a model four-helix bundle protein, HP7. Both the average ensemble secondary structure and types of local disorder were monitored, without perturbation of the solvent, pH, or temperature. The molten globule to ordered transition is induced by stepwise coordination of two heme molecules. Persistent dUVRR spectral features in the amide III region at 1295–1301 and 1335–1338 cm−1 confirm previous observations that HP7 remains predominantly helical in the molten globule versus the fully ordered state. Additionally, these spectra represent the first demonstration of conserved helical content in a molten globule protein. With successive heme binding significant losses are observed in the spectral intensity of the amide III3 and S regions (1230–1260 and 1390 cm−1, respectively), which are known to be sensitive to local disorder. These observations indicate that there is a decrease in the structural populations able to explore various extended conformations, with successive heme binding events. DUVRR spectra indicate that the first heme coordination between two helical segments diminishes exploration of more elongated backbone structural

  12. Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins

    PubMed Central

    Wen, Jie; Wu, Jihui; Zhou, Pei

    2011-01-01

    Intrinsically disordered proteins (IDPs) play important roles in many critical cellular processes. Due to their limited chemical shift dispersion, IDPs often require four pairs of resonance connectivities (Hα, Cα, Cβ and CO) for establishing sequential backbone assignment. Because most conventional 4-D triple-resonance experiments share an overlapping Cα evolution period, combining existing 4-D experiments does not offer an optimal solution for non-redundant collection of a complete set of backbone resonances. Using alternative chemical shift evolution schemes, we propose a new pair of 4-D triple resonance experiments—HA(CA)CO(CA)NH/HA(CA)CONH—that complement the 4-D HNCACB/HN(CO)CACB experiments to provide complete backbone resonance information. Collection of high-resolution 4-D spectra with sparse sampling and FFT-CLEAN processing enables efficient acquisition and assignment of complete backbone resonances of IDPs. Importantly, because the CLEAN procedure iteratively identifies resonance signals and removes their associating aliasing artifacts, it greatly reduces the dependence of the reconstruction quality on sampling schemes and produces high-quality spectra even with less-than-optimal sampling schemes. PMID:21277815

  13. 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

  14. 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

  15. 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

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

    PubMed

    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.

  17. 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

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

    PubMed

    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

  19. Metamorphic proteins mediate evolutionary transitions of structure.

    PubMed

    Yadid, Itamar; Kirshenbaum, Noam; Sharon, Michal; Dym, Orly; Tawfik, Dan S

    2010-04-20

    The primary sequence of proteins usually dictates a single tertiary and quaternary structure. However, certain proteins undergo reversible backbone rearrangements. Such metamorphic proteins provide a means of facilitating the evolution of new folds and architectures. However, because natural folds emerged at the early stages of evolution, the potential role of metamorphic intermediates in mediating evolutionary transitions of structure remains largely unexplored. We evolved a set of new proteins based on approximately 100 amino acid fragments derived from tachylectin-2--a monomeric, 236 amino acids, five-bladed beta-propeller. Their structures reveal a unique pentameric assembly and novel beta-propeller structures. Although identical in sequence, the oligomeric subunits adopt two, or even three, different structures that together enable the pentameric assembly of two propellers connected via a small linker. Most of the subunits adopt a wild-type-like structure within individual five-bladed propellers. However, the bridging subunits exhibit domain swaps and asymmetric strand exchanges that allow them to complete the two propellers and connect them. Thus, the modular and metamorphic nature of these subunits enabled dramatic changes in tertiary and quaternary structure, while maintaining the lectin function. These oligomers therefore comprise putative intermediates via which beta-propellers can evolve from smaller elements. Our data also suggest that the ability of one sequence to equilibrate between different structures can be evolutionary optimized, thus facilitating the emergence of new structures.

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

    DOE PAGES

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

    2016-02-15

    In this paper, 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 bymore » 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. In conclusion, these general trends of dihedral conformational transitions provide important insights into the hierarchical dynamics and complex free energy landscapes of functional proteins.« less

  1. 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

  2. Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

    PubMed Central

    Tortora, Giampaolo; Caputo, Rosa; Damiano, Vincenzo; Bianco, Roberto; Pepe, Stefano; Bianco, A. Raffaele; Jiang, Zhiwei; Agrawal, Sudhir; Ciardiello, Fortunato

    1997-01-01

    Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs. PMID:9356493

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

    PubMed

    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 (15)N-labeled SH3 (Src homology 3) domain proteins in aqueous buffer were used to generate general order parameters (S(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(2)) were derived from the MD trajectory. Correlation analysis using the Gromos force field indicated that S(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(2) parameters from MD simulations with charges for all three histidines and with the SPC/E water model correlated well with S(2) calculated from the experimental NMR relaxation measurements, in a site-specific manner.

  4. A Bayesian-probability-based method for assigning protein backbone dihedral angles based on chemical shifts and local sequences.

    PubMed

    Wang, Jun; Liu, Haiyan

    2007-01-01

    Chemical shifts contain substantial information about protein local conformations. We present a method to assign individual protein backbone dihedral angles into specific regions on the Ramachandran map based on the amino acid sequences and the chemical shifts of backbone atoms of tripeptide segments. The method uses a scoring function derived from the Bayesian probability for the central residue of a query tripeptide segment to have a particular conformation. The Ramachandran map is partitioned into representative regions at two levels of resolution. The lower resolution partitioning is equivalent to the conventional definitions of different secondary structure regions on the map. At the higher resolution level, the alpha and beta regions are further divided into subregions. Predictions are attempted at both levels of resolution. We compared our method with TALOS using the original TALOS database, and obtained comparable results. Although TALOS may produce the best results with currently available databases which are much enlarged, the Bayesian-probability-based approach can provide a quantitative measure for the reliability of predictions.

  5. 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.

  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. Unraveling the complexity of protein backbone dynamics with combined (13)C and (15)N solid-state NMR relaxation measurements.

    PubMed

    Lamley, Jonathan M; Lougher, Matthew J; Sass, Hans Juergen; Rogowski, Marco; Grzesiek, Stephan; Lewandowski, Józef R

    2015-09-14

    Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce (13)C spin-lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of (13)C'R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz (1)H Larmor frequencies and compare them to (13)C'R1, (15)N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when (15)N data is used alone. We also discuss how internal motions characterized by different time scales contribute to (15)N and (13)C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution.

  8. Predicting backbone Cα angles and dihedrals from protein sequences by stacked sparse auto-encoder deep neural network.

    PubMed

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

    2014-10-30

    Because a nearly constant distance between two neighbouring Cα atoms, local backbone structure of proteins can be represented accurately by the angle between C(αi-1)-C(αi)-C(αi+1) (θ) and a dihedral angle rotated about the C(αi)-C(αi+1) bond (τ). θ and τ angles, as the representative of structural properties of three to four amino-acid residues, offer a description of backbone conformations that is complementary to φ and ψ angles (single residue) and secondary structures (>3 residues). Here, we report the first machine-learning technique for sequence-based prediction of θ and τ angles. Predicted angles based on an independent test have a mean absolute error of 9° for θ and 34° for τ with a distribution on the θ-τ plane close to that of native values. The average root-mean-square distance of 10-residue fragment structures constructed from predicted θ and τ angles is only 1.9Å from their corresponding native structures. Predicted θ and τ angles are expected to be complementary to predicted ϕ and ψ angles and secondary structures for using in model validation and template-based as well as template-free structure prediction. The deep neural network learning technique is available as an on-line server called Structural Property prediction with Integrated DEep neuRal network (SPIDER) at http://sparks-lab.org.

  9. Root-mean-square-deviation-based rapid backbone resonance assignments in proteins.

    PubMed

    Rout, Ashok K; Barnwal, Ravi P; Agarwal, Geetika; Chary, Kandala V R

    2010-10-01

    We have shown that the methodology based on the estimation of root-mean-square deviation (RMSD) between two sets of chemical shifts is very useful to rapidly assign the spectral signatures of (1)H(N), (13)C(α), (13)C(β), (13)C', (1)H(α) and (15)N spins of a given protein in one state from the knowledge of its resonance assignments in a different state, without resorting to routine established procedures (manual and automated). We demonstrate the utility of this methodology to rapidly assign the 3D spectra of a metal-binding protein in its holo-state from the knowledge of its assignments in apo-state, the spectra of a protein in its paramagnetic state from the knowledge of its assignments in diamagnetic state and, finally, the spectra of a mutant protein from the knowledge of the chemical shifts of the corresponding wild-type protein. The underlying assumption of this methodology is that, it is impossible for any two amino acid residues in a given protein to have all the six chemical shifts degenerate and that the protein under consideration does not undergo large conformational changes in going from one conformational state to another. The methodology has been tested using experimental data on three proteins, M-crystallin (8.5 kDa, predominantly β-sheet, for apo- to holo-state), Calbindin (7.5 kDa, predominantly α-helical, for diamagnetic to paramagnetic state and apo to holo) and EhCaBP1 (14.3 kDa, α-helical, the wild-type protein with one of its mutant). In all the cases, the extent of assignment is found to be greater than 85%.

  10. Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

    PubMed

    Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2014-10-01

    Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

  11. 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.

  12. 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

  13. Protein structure quality assessment based on the distance profiles of consecutive backbone Cα atoms.

    PubMed

    Chakraborty, Sandeep; Venkatramani, Ravindra; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya M

    2013-01-01

    Predicting the three dimensional native state structure of a protein from its primary sequence is an unsolved grand challenge in molecular biology. Two main computational approaches have evolved to obtain the structure from the protein sequence - ab initio/de novo methods and template-based modeling - both of which typically generate multiple possible native state structures. Model quality assessment programs (MQAP) validate these predicted structures in order to identify the correct native state structure. Here, we propose a MQAP for assessing the quality of protein structures based on the distances of consecutive Cα atoms. We hypothesize that the root-mean-square deviation of the distance of consecutive Cα (RDCC) atoms from the ideal value of 3.8 Å, derived from a statistical analysis of high quality protein structures (top100H database), is minimized in native structures. Based on tests with the top100H set, we propose a RDCC cutoff value of 0.012 Å, above which a structure can be filtered out as a non-native structure. We applied the RDCC discriminator on decoy sets from the Decoys 'R' Us database to show that the native structures in all decoy sets tested have RDCC below the 0.012 Å cutoff. While most decoy sets were either indistinguishable using this discriminator or had very few violations, all the decoy structures in the fisa decoy set were discriminated by applying the RDCC criterion. This highlights the physical non-viability of the fisa decoy set, and possible issues in benchmarking other methods using this set. The source code and manual is made available at https://github.com/sanchak/mqap and permanently available on 10.5281/zenodo.7134.

  14. 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

  15. Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins.

    PubMed

    Bagai, Ireena; Ragsdale, Stephen W; Zuiderweg, Erik R P

    2011-02-01

    The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve (15)N and (1)HN resonances. The assignment becomes problematic when there is resonance overlap of (15)N-(1)HN cross peaks. For such residues, one cannot unambiguously link the "left" side of the NH root to the "right" side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a solution to this problem: a hybrid (4d,3d) reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance is modulated with the frequency of the Ca(i-1) resonance, which helps in resolving the ambiguity involved in connecting the Ca(i) and Ca(i-1) resonances for overlapping NH roots. The experiment has limited sensitivity, and is only suited for small or unfolded proteins. In a companion experiment, (4d,3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(i-1) resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(i-1) resonances for overlapping NH roots. PMID:21190062

  16. Inter-helical interactions in membrane proteins: analysis based on the local backbone geometry and the side chain interactions.

    PubMed

    Jha, Anupam Nath; Vishveshwara, Saraswathi

    2009-06-01

    The availability of a significant number of the structures of helical membrane proteins has prompted us to investigate the mode of helix-helix packing. In the present study, we have considered a dataset of alpha-helical membrane proteins representing structures solved from all the known superfamilies. We have described the geometry of all the helical residues in terms of local coordinate axis at the backbone level. Significant inter-helical interactions have been considered as contacts by weighing the number of atom-atom contacts, including all the side-chain atoms. Such a definition of local axis and the contact criterion has allowed us to investigate the inter-helical interaction in a systematic and quantitative manner. We show that a single parameter (designated as alpha), which is derived from the parameters representing the mutual orientation of local axes, is able to accurately capture the details of helix-helix interaction. The analysis has been carried out by dividing the dataset into parallel, anti-parallel, and perpendicular orientation of helices. The study indicates that a specific range of alpha value is preferred for interactions among the anti-parallel helices. Such a preference is also seen among interacting residues of parallel helices, however to a lesser extent. No such preference is seen in the case of perpendicular helices, the contacts that arise mainly due to the interaction of surface helices with the end of the trans-membrane helices. The study supports the prevailing view that the anti-parallel helices are well packed. However, the interactions between helices of parallel orientation are non-trivial. The packing in alpha-helical membrane proteins, which is systematically and rigorously investigated in this study, may prove to be useful in modeling of helical membrane proteins.

  17. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

    PubMed

    Choi, Yong-Geun; Park, Chin-Ju; Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa

    2015-02-01

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  18. Using MUSIC and CC(CO)NH for backbone assignment of two medium-sized proteins not fully accessible to standard 3D NMR.

    PubMed

    Brenner, Annette K; Frøystein, Nils Åge

    2014-01-01

    The backbone assignment of medium-sized proteins is rarely as straightforward as that of small proteins, and thus often requires creative solutions. Here, we describe the application of a combination of standard 3D heteronuclear methods with CC(CO)NH and a variety of MUltiplicity Selective In-phase Coherence transfer (MUSIC) experiments. Both CC(CO)NH and MUSIC are, in theory, very powerful methods for the backbone assignment of proteins. Due to low sensitivity, their use has usually been linked to small proteins only. However, we found that combining CC(CO)NH and MUSIC experiments simplified the assignment of two challenging medium-sized proteins of 13 and 19.5 kDa, respectively. These methods are to some extent complementary to each other: CC(CO)NH acquired with a long isotropic mixing time can identify amino acids with large aliphatic side chains. Whereas the most sensitive MUSIC experiments identify amino acid types that cannot be detected by CC(CO)NH, comprising the residues with acid and amide groups, and aromatic rings in their side chains. Together these methods provide a means of identifying the majority of peaks in the 2D 15N HSQC spectrum which simplifies the backbone assignment work even for proteins, e.g., small kinases, whose standard spectra resulted in little spectral resolution and low signal intensities.

  19. Significant role of the DNA backbone in mediating the transition origin of electronic excitations of B-DNA - implication from long range corrected TDDFT and quantified NTO analysis

    NASA Astrophysics Data System (ADS)

    Li, Jian-Hao; Chai, Jeng-Da; Guo, Guang-Yu; Hayashi, Michitoshi

    We systematically investigate the possible complex transition origin of electronic excitations of giant molecular systems by using the recently proposed QNTO analysis [J.-H. Li, J.-D. Chai, G. Y. Guo and M. Hayashi, Chem. Phys. Lett., 2011, 514, 362.] combined with long-range corrected TDDFT calculations. Thymine (Thy) related excitations of biomolecule B-DNA are then studied as examples, where the model systems have been constructed extracting from the perfect or a X-ray crystal (PDB code 3BSE) B-DNA structure with at least one Thy included. In the first part, we consider the systems composed of a core molecular segment (e.g. Thy, di-Thy) and a surrounding physical/chemical environment of interest (e.g. backbone, adjacent stacking nucleobases) and examine how the excitation properties of the core vary in response to the environment. We find that the orbitals contributed from DNA backbone and surrounding nucleobases often participate in a transition of Thy-related excitations affecting their composition, absorption energy, and oscillator strength. In the second part, we take into account geometrically induced variation of the excitation properties of various B-DNA segments, e.g. di-Thy, dTpdT etc., obtained from different sources (ideal and 3BSE). It is found that the transition origin of several Thy-related excitations of these segments is sensitive to slight conformational variations, suggesting that DNA with thermal motions in cells may from time to time exhibit very different photo-induced physical and/or chemical processes.

  20. 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

  1. Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools.

    PubMed

    Monleón, Daniel; Colson, Kimberly; Moseley, Hunter N B; Anklin, Clemens; Oswald, Robert; Szyperski, Thomas; Montelione, Gaetano T

    2002-01-01

    Rapid data collection, spectral referencing, processing by time domain deconvolution, peak picking and editing, and assignment of NMR spectra are necessary components of any efficient integrated system for protein NMR structure analysis. We have developed a set of software tools designated AutoProc, AutoPeak, and AutoAssign, which function together with the data processing and peak-picking programs NMRPipe and Sparky, to provide an integrated software system for rapid analysis of protein backbone resonance assignments. In this paper we demonstrate that these tools, together with high-sensitivity triple resonance NMR cryoprobes for data collection and a Linux-based computer cluster architecture, can be combined to provide nearly complete backbone resonance assignments and secondary structures (based on chemical shift data) for a 59-residue protein in less than 30 hours of data collection and processing time. In this optimum case of a small protein providing excellent spectra, extensive backbone resonance assignments could also be obtained using less than 6 hours of data collection and processing time. These results demonstrate the feasibility of high throughput triple resonance NMR for determining resonance assignments and secondary structures of small proteins, and the potential for applying NMR in large scale structural proteomics projects.

  2. Backbone 1H, 13C, and 15N NMR assignments for the Cyanothece 51142 protein cce_0567: a protein associated with nitrogen fixation in the DUF683 family

    SciTech Connect

    Buchko, Garry W.; Sofia, Heidi J.

    2008-06-01

    The recently sequenced genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 (contig 83.1_1_243_746) contains the sequence for an hypothetical protein that falls into the DUF683 family. As observed for the other 54 DUF683 proteins currently listed in the GenBank database, this 78-residue (9.0 kDa) protein in Cyanothece is also found in a nitrogen fixation gene cluster suggesting that it is involved in the process. To date no structural information exists for any of the proteins in the DUF683 family. In an effort to elucidate the biochemical role DUF683 may play in nitrogen fixation and to obtain structural information for a member of the DUF683 protein family, a construct containing DUF683 from Cyanothece 51142 was generated, expressed, purified, and the solution properties characterized. A total rotational correlation time (tc) of 17.1 ns was estimated by nuclear magnetic resonance (NMR) spectroscopy suggesting a molecular weight of ~ 40 kDa, an observation dictating that DUF683 is a tetramer in solution. Using triple-labeled (2H, 13C, 15N) and residue-specific 15N-labeled amino acids (L, K, V, and E/Q) samples, most of the backbone and side chain resonances for DUF683 were assigned. The 13C alpha chemical shifts and NOESY NMR data indicate that the protein is helical from K18-E75.

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

    PubMed Central

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

    2015-01-01

    About 8,000 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

  4. 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

  5. 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

    Faraggi, Eshel; Xue, Bin; Zhou, Yaoqi

    2009-03-01

    This article 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 10-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 degrees for psi, and 22 degrees for phi. The method is available as a Real-SPINE 3.0 server in http://sparks.informatics.iupui.edu.

  6. 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.

  7. Backbone ¹H, ¹³C and ¹⁵N resonance assignments of the α-helical membrane protein TM0026 from Thermotoga maritima.

    PubMed

    Kroncke, Brett M; Columbus, Linda

    2013-10-01

    Critical to the use of solution NMR to describe the structure and flexibility of membrane proteins is the thorough understanding of the degree of perturbation induced by the detergent or other membrane mimetic. To develop a deeper understanding of the interaction between membrane proteins and micelles or bicelles, we will investigate the differences in structure and flexibility of a model membrane protein TM0026 from Thermotoga maritima using solution NMR. A comparison of the structural differences between TM0026 solubilized in different detergent combinations will provide important insight into the degree of modulation of membrane proteins by detergent physical properties. Here we report the nearly complete backbone and Cβ resonance assignments of the two transmembrane helical model protein TM0026. These assignments are the first step to using TM0026 to elucidate the interaction between membrane proteins and membrane mimetics.

  8. Backbone dynamics of a model membrane protein: 13C NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein.

    PubMed

    Henry, G D; Weiner, J H; Sykes, B D

    1986-02-11

    The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection. 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles. A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of [3-13C]alanine. Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total). Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different. The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems. The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure. Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e. The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. 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

  10. Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

    PubMed

    Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

    2004-12-01

    Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

  11. Backbone dynamics in collagen

    NASA Astrophysics Data System (ADS)

    Aliev, Abil E.

    2004-11-01

    Peptide backbone motions of collagen have been extensively studied in the past. The experimental results were interpreted using a model of a collagen rod librating about its helix axis. Considering the size of the collagen molecule and the presence of cross-linked molecules, motional amplitudes derived for the helix axis libration were unusually high. Using solid-state NMR 13C chemical shift anisotropy and 2H quadrupolar lineshape analysis for five different isotope labelled collagens we show that motional averaging of the NMR interactions occurs primarily via small-angle librations about internal bond directions. This type of dynamics is compatible with both the presence of cross-links in collagen and the X-ray data, as well as dynamic models used for other proteins.

  12. 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.

  13. 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.

  14. Modeling the "glass" transition in proteins.

    PubMed

    Sitnitsky, A E

    2002-02-01

    A model of a protein as a disordered system of identical spherical particles (which imitate protein side chains) interacting with each other via a repulsive soft sphere potential U(r) infinity r(-beta) is constructed. The particles undergo the conformational motion (CM) within their own harmonic conformational potentials around some mean equilibrium positions ascribed by the tertiary structure of the protein. A first principles calculation of the positional correlation functions for the CM is carried out. The general analysis is exemplified by the case in which the mean equilibrium positions of the particles form a cubic tightly-packed (face- centered) lattice (each particle has 12 nearest neighbors) with the step b(hydr) =6.6 A (the average distance between the centers of mass of hydrated protein subunits). The model yields dramatic slowing down of the relaxation with the decrease of temperature followed by a sharp glass transition at some crossover temperature T(c) < 200 K. At the transition the liquid-like dynamic behavior (the correlation functions tend to zero with time) is altered by the glass-like one (the correlation functions tend with time to some non-zero limit). In the liquid-like region above the crossover temperature the relaxation exhibits distinct alpha-process following the beta-one. The glass transition results from the interaction of the particles. Thus the model suggests that namely direct interactions of the fragments of protein structure rather than protein-solvent interactions are the origin of the phenomenon of the glass transition. The known increase of T(c) up to 300 K at dehydration of the protein is attributed to the known concomitant compression of the globule upon drying by about 4-6% so that positions of individual atoms displace by about 0.6 A (modeled by the decrease of the step of the lattice b by 0.6 A so that b(dehydr)=6 A). The model suggests that the solvent influences the phenomenon of the glass transition indirectly

  15. Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris.

    PubMed

    Aramini, James M; Hamilton, Keith; Rossi, Paolo; Ertekin, Asli; Lee, Hsiau-Wei; Lemak, Alexander; Wang, Huang; Xiao, Rong; Acton, Thomas B; Everett, John K; Montelione, Gaetano T

    2012-05-01

    Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro.

  16. Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris.

    PubMed

    Aramini, James M; Hamilton, Keith; Rossi, Paolo; Ertekin, Asli; Lee, Hsiau-Wei; Lemak, Alexander; Wang, Huang; Xiao, Rong; Acton, Thomas B; Everett, John K; Montelione, Gaetano T

    2012-05-01

    Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro. PMID:22497251

  17. NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition.

    PubMed

    Mukherjee, Munia; Dutta, Kaushik; White, Mark A; Cowburn, David; Fox, Robert O

    2006-06-01

    Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered to be the primary viral determinant involved in the virus-host-cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4 viruses in the serogroup. We have determined the solution structure of LGT-E-D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT-E-D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.

  18. 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

  19. 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 Central

    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

  20. 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

  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. Backbone and side-chain chemical shift assignments for the C-terminal domain of Tcb2, a cytoskeletal calcium-binding protein from Tetrahymena thermophila.

    PubMed

    Kilpatrick, Adina M; Gurrola, Theodore E; Sterner, Robert C; Sleister, Heidi M; Honts, Jerry E; Fowler, C Andrew

    2016-10-01

    Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.

  3. 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

  4. A ‘just-in-time’ HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity

    NASA Astrophysics Data System (ADS)

    Werner-Allen, Jon W.; Jiang, Ling; Zhou, Pei

    2006-07-01

    Among the suite of commonly used backbone experiments, HNCACO presents an unresolved sensitivity limitation due to fast 13CO transverse relaxation and passive 13Cα-13Cβ coupling. Here, we present a high-sensitivity 'just-in-time' (JIT) HN(CA)CO pulse sequence that uniformly refocuses 13Cα-13Cβ coupling while collecting 13CO shifts in real time. Sensitivity comparisons of the 3-D JIT HN(CA)CO, a CT-HMQC-based control, and a HSQC-based control with selective 13Cα inversion pulses were performed using a 2H/13C/15N labeled sample of the 29 kDa HCA II protein at 15 °C. The JIT experiment shows a 42% signal enhancement over the CT-HMQC-based experiment. Compared to the HSQC-based experiment, the JIT experiment is 16% less sensitive for residues experiencing proper 13Cα refocusing and 13Cα-13Cβ decoupling. However, for the remaining residues, the JIT spectrum shows a 106% average sensitivity gain over the HSQC-based experiment. The high-sensitivity JIT HNCACO experiment should be particularly beneficial for studies of large proteins to provide 13CO resonance information regardless of residue type.

  5. Strike a balance: optimization of backbone torsion parameters of AMBER polarizable force field for simulations of proteins and peptides.

    PubMed

    Wang, Zhi-Xiang; Zhang, Wei; Wu, Chun; Lei, Hongxing; Cieplak, Piotr; Duan, Yong

    2006-04-30

    Based on the AMBER polarizable model (ff02), we have re-optimized the parameters related to the main-chain (Phi, Psi) torsion angles by fitting to the Boltzmann-weighted average quantum mechanical (QM) energies of the important regions (i.e., beta, P(II), alpha(R), and alpha(L) 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 beta, P(II) and alpha(R) 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.

  6. Intra-residue interactions in proteins: interplay between serine or cysteine side chains and backbone conformations, revealed by laser spectroscopy of isolated model peptides.

    PubMed

    Alauddin, Mohammad; Biswal, Himansu S; Gloaguen, Eric; Mons, Michel

    2015-01-21

    Intra-residue interactions play an important role in proteins by influencing local folding of the backbone. Taking advantage of the capability of gas phase experiments to provide relevant information on the intrinsic H-bonding pattern of isolated peptide chains, the intra-residue interactions of serine and cysteine residues, i.e., OH/SH···OC(i) C6 and NH(i···)O/S C5 interactions in Ser/Cys residues, are probed by laser spectroscopy of isolated peptides. The strength of these local side chain-main chain interactions, elegantly documented from their IR spectral features for well-defined conformations of the main chain, demonstrates that a subtle competition exists between the two types of intra-residue bond: the C6 H-bond is the major interaction with Ser, in contrast to Cys where C5 interaction takes over. The restricted number of conformers observed in the gas phase experiment with Ser compared to Cys (where both extended and folded forms are observed) also suggests a significant mediation role of these intra-residue interactions on the competition between the several main chain folding patterns. PMID:25482851

  7. Protein dynamical transition at 110 K

    PubMed Central

    Kim, Chae Un; Tate, Mark W.; Gruner, Sol M.

    2011-01-01

    Proteins are known to undergo a dynamical transition at around 200 K but the underlying mechanism, physical origin, and relationship to water are controversial. Here we report an observation of a protein dynamical transition as low as 110 K. This unexpected protein dynamical transition precisely correlated with the cryogenic phase transition of water from a high-density amorphous to a low-density amorphous state. The results suggest that the cryogenic protein dynamical transition might be directly related to the two liquid forms of water proposed at cryogenic temperatures. PMID:22167801

  8. Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment.

    PubMed

    Chen, Kang; Tjandra, Nico

    2009-03-01

    Protein backbone (15)N spin relaxation rates measured by solution NMR provide useful dynamic information with a site-specific resolution. The conventional method is to record a series of 2D (1)H-(15)N HSQC spectra with varied relaxation delays, and derive relaxation rate from the following curve fitting on the resonance intensities. Proteins with poorly resolved spectra often require several 3D HNCO spectra to be collected on a (15)N/(13)C double labeled protein sample. In order to reduce the relaxation dimension Carr et al. (P.A. Carr, D.A. Fearing, A.G. Palmer, 3D accordion spectroscopy for measuring N-15 and (CO)-Carbon-13 relaxation rates in poorly resolved NMR spectra, J. Magn. Reson. 132 (1998) 25-33) employed an Accordion type HNCO pulse sequence to obtain (15)N or (13)C T(1) relaxation rates by numerical fitting of the relaxation interfered free induction decay (FID) data. To avoid intensive analysis of the time domain data, we propose a modified protocol to measure (15)N T(1) and T(2) relaxation rates from easily obtained line-widths in an Accordion HNCO spectrum. Both T(1) and T(2) relaxation could be simultaneously convoluted into the constant-time evolution periods of (13)C' and (15)N, respectively. The relaxation delay was allowed to reach at least 3 x T(1) or 3 x T(2) so that the signal was substantially decayed by the end of the FID, and the resulting peak full-width at half height (FWHH) could be directly used to calculate relaxation rate. When applied to the 76-residue Ubiquitin and the 226-residue glutamine-binding protein (GlnBP), this method yielded T(1) and T(2) values deviating on average by 4-6% and 5-7%, respectively, from the measurements based on the conventional 2D method. In comparison, the conventional methods possessed intrinsic error ranges of 2-4% for T(1) and 3-6% for T(2). In addition to comparable accuracy, the fully-relaxed Accordion HNCO method presented here allowed measurements of relaxation rates for resonances unresolved in

  9. Dynamic transition associated with the thermal denaturation of a small Beta protein.

    PubMed Central

    Russo, Daniela; Pérez, Javier; Zanotti, Jean-Marc; Desmadril, Michel; Durand, Dominique

    2002-01-01

    We studied the temperature dependence of the picosecond internal dynamics of an all-beta protein, neocarzinostatin, by incoherent quasielastic neutron scattering. Measurements were made between 20 degrees C and 71 degrees C in heavy water solution. At 20 degrees C, only 33% of the nonexchanged hydrogen atoms show detectable dynamics, a number very close to the fraction of protons involved in the side chains of random coil structures, therefore suggesting a rigid structure in which the only detectable diffusive movements are those involving the side chains of random coil structures. At 61.8 degrees C, although the protein structure is still native, slight dynamic changes are detected that could reflect enhanced backbone and beta-sheet side-chain motions at this higher temperature. Conversely, all internal dynamics parameters (amplitude of diffusive motions, fraction of immobile scatterers, mean-squared vibration amplitude) rapidly change during heat-induced unfolding, indicating a major loss of rigidity of the beta-sandwich structure. The number of protons with diffusive motion increases markedly, whereas the volume occupied by the diffusive motion of protons is reduced. At the half-transition temperature (T = 71 degrees C) most of backbone and beta-sheet side-chain hydrogen atoms are involved in picosecond dynamics. PMID:12414711

  10. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    SciTech Connect

    Shi, Pan; Xi, Zhaoyong; Wang, Hu; Shi, Chaowei; Xiong, Ying; Tian, Changlin

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  11. Dynamics and dynamical transitions in proteins

    NASA Astrophysics Data System (ADS)

    Vurnal, Derya

    Neutron scattering experiments and molecular dynamics simulations are the most effective tools to explore the dynamics of hydrogen in proteins. The mean square displacement (MSD) of hydrogen (H) in proteins has been extensively measured using neutron scattering and calculated using molecular dynamics simulations. A small MSD is observed at low temperatures and the slope of the MSD significantly increases at a specific temperature TD. This increase in the slope of the MSD is identified as a dynamical transition, and the temperature it takes place at a specific temperature which is denoted a dynamical transition temperature T D. The observed MSD in neutron scattering experiments depend on the energy resolution of the instrument. In this thesis, we first focus on the resolution dependent of the observed MSD exp in neutron scattering experiments. We propose a method for obtaining the intrinsic MSD exp of H, which is independent of the resolution of the instrument employed, in proteins. The intrinsic MSD is defined as the infinite time value of MSD which appears in the well-known Debye-Waller factor. In this method, a model of the resolution broadened elastic incoherent dynamic structure factor SR(Q, o = 0) is developed to extract the intrinsic MSD from the resolution dependent data. The model contains the intrinsic MSD, the instrument resolution width and a relaxation frequency characterizing the motions of H in proteins. The model of SR(Q, o = 0) is fitted to the resolution broadened DSF data already published in the recent literature and the intrinsic MSD in three proteins was successfully obtained. Later, we constructed a model for the incoherent intermediate scattering function I(Q, t) to obtain the intrinsic, long-time MSD of H in proteins from finite time molecular dynamics simulations. In the literature, the simulated MSD increases with increasing time and does not reach a certain limiting value at even 10 ns. The infinite time MSD, , is the long

  12. 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

  13. Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers.

    PubMed Central

    Song, Z; Antzutkin, O N; Lee, Y K; Shekar, S C; Rupprecht, A; Levitt, M H

    1997-01-01

    Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 14 PMID:9284321

  14. Instantaneous Normal Modes and the Protein Glass Transition

    PubMed Central

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

    2009-01-01

    Abstract 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. PMID:19167298

  15. 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.

  16. Photocleavage of the Polypeptide Backbone by 2-Nitrophenylalanine

    PubMed Central

    Peters, Francis B.; Brock, Ansgar; Wang, Jiangyun; Schultz, Peter G.

    2009-01-01

    Summary Photocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site specifically photocleaved. PMID:19246005

  17. Backbone and side chain NMR assignment, along with the secondary structure prediction of RRM2 domain of La protein from a lower eukaryote exhibiting identical structural organization with its human homolog.

    PubMed

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

    2015-04-01

    The La protein (Lupus antigen), a key mediator during biogenesis of RNA polymerase III transcripts, contains a characteristic La motif and one or two RNA recognition motif (RRM) domains, depending on the organism of origin. The RRM1 domain is conserved in higher eukaryotes and located in the N-terminal region, whereas the C-terminal RRM2 domain is absent in most lower eukaryotes and its specific role remains, so far, uncharacterized. Here, we present the backbone and side-chain assignment of the (1)H, (13)C and (15)N resonances of RRM2 of La protein from Dictyostelium discoideum. Interestingly, the La protein in this lower eukaryote, exhibits high homology to its human counterpart. Moreover, it contains two RRM domains, instead of one, raising questions on its evolutionary origin and the putative role of RRM2 in vivo. We also provide its secondary structure as predicted by the TALOS+ online tool.

  18. 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

  19. 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

  20. The myosin filament XIV backbone structure.

    PubMed Central

    Ashton, F T; Weisel, J; Pepe, F A

    1992-01-01

    The substructure of the thick filaments of chemically skinned chicken pectoralis muscle was investigated by electron microscopy. Images of transverse sections of the myosin filaments were determined to have threefold symmetry by cross-correlation analysis, which gives an unbiased determination of the rotational symmetry of the images. Resolution, using the phase residual test (Frank et al. 1981. Science [Wash. DC]. 214:1353-1355), was found to be between 3.2 and 3.6 nm. Three arrangements of nine subfilaments in the backbone were found in all regions of the filament at ionic strengths of 20 and 200 mM. In the average images of two of these, there were three dense central subfilaments and three pairs of subfilaments on the surface of the thick filament. In the average image of the third arrangement, all of the protein mass of the nine subfilaments was on the surface of the filament with three of them showing less variation in position than the others. A fourth arrangement appearing to be transitional between two of these was seen often at 200 mM ionic strength and only rarely at 20 mM. On average, the myosin subfilaments were parallel to the long axis of the filament. The different arrangements of subfilaments appear to be randomly distributed among the filaments in a transverse section of the A-band. Relative rotational orientations with respect to the hexagonal filament lattice, using the three densest subfilaments as reference showed a major clustering (32%) of filaments within one 10 degrees spread, a lesser clustering (15%) at 90 degrees to the first, and the remainder scattered thinly over the rest of the 120 degrees range. There was no obvious pattern of distribution of the two predominant orientations that could define a superlattice in the filament lattice. Images FIGURE 2 FIGURE 6 FIGURE 8 PMID:1617136

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Genomic and expression analysis of transition proteins in Drosophila.

    PubMed

    Alvi, Zain A; Chu, Tin-Chun; Schawaroch, Valerie; Klaus, Angela V

    2015-01-01

    The current study was aimed at analyzing putative protein sequences of the transition protein-like proteins in 12 Drosophila species based on the reference sequences of transition protein-like protein (Tpl (94D) ) expressed in Drosophila melanogaster sperm nuclei. Transition proteins aid in transforming chromatin from a histone-based nucleosome structure to a protamine-based structure during spermiogenesis - the post-meiotic stage of spermatogenesis. Sequences were obtained from NCBI Ref-Seq database using NCBI ORF-Finder (PSI-BLAST). Sequence alignments and analysis of the amino acid content indicate that orthologs for Tpl (94D) are present in the melanogaster species subgroup (D. simulans, D. sechellia, D. erecta, and D. yakuba), D. ananassae, and D. pseudoobscura, but absent in D. persmilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi. Transcriptome next generation sequence (RNA-Seq) data for testes and ovaries was used to conduct differential gene expression analysis for Tpl (94D) in D. melanogaster, D. simulans, D. yakuba, D. ananassae, and D. pseudoobscura. The identified Tpl (94D) orthologs show high expression in the testes as compared to the ovaries. Additionally, 2 isoforms of Tpl (94D) were detected in D. melanogaster with isoform A being much more highly expressed than isoform B. Functional analyses of the conserved region revealed that the same high mobility group (HMG) box/DNA binding region is conserved for both Drosophila Tpl (94D) and Drosophila protamine-like proteins (MST35Ba and MST35Bb). Based on the rigorous bioinformatic approach and the conservation of the HMG box reported in this work, we suggest that the Drosophila Tpl (94D) orthologs should be classified as their own transition protein group.

  7. 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

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

    NASA Astrophysics Data System (ADS)

    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.

  9. 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

  10. The dominant folding route minimizes backbone distortion in SH3.

    PubMed

    Lammert, Heiko; Noel, Jeffrey K; Onuchic, José N

    2012-01-01

    Energetic frustration in protein folding is minimized by evolution to create a smooth and robust energy landscape. As a result the geometry of the native structure provides key constraints that shape protein folding mechanisms. Chain connectivity in particular has been identified as an essential component for realistic behavior of protein folding models. We study the quantitative balance of energetic and geometrical influences on the folding of SH3 in a structure-based model with minimal energetic frustration. A decomposition of the two-dimensional free energy landscape for the folding reaction into relevant energy and entropy contributions reveals that the entropy of the chain is not responsible for the folding mechanism. Instead the preferred folding route through the transition state arises from a cooperative energetic effect. Off-pathway structures are penalized by excess distortion in local backbone configurations and contact pair distances. This energy cost is a new ingredient in the malleable balance of interactions that controls the choice of routes during protein folding.

  11. hnCOcaNH and hncoCANH pulse sequences for rapid and unambiguous backbone assignment in (13C, 15N) labeled proteins.

    PubMed

    Kumar, Dinesh; Reddy, Jithender G; Hosur, Ramakrishna V

    2010-09-01

    Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the context of structural proteomics research. The conventional HNCA and HN(CA)CO pulse sequences, routinely used for sequential backbone assignment, have the limitation that they cannot distinguish inter- and intra-residue correlations. In order to remove this ambiguity, one has to record HNCO and HN(CO)CA or sequential HNCA experiments which provide unambiguous information of sequential correlations. However, this almost doubles the experimental time. Besides, they require repeated scanning through the (15)N planes to search for the matching peaks along the carbon dimension. In this background, we present here two pulse sequences, termed as hncoCANH and hnCOcaNH that lead to spectra equivalent to HNCA and HN(CA)CO spectra, respectively, but with direct distinction of inter- and intra-residue peaks; these occur with opposite signs in the new experiments. The two pulse sequences have been derived by simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] to frequency-label (13)C(alpha) or (13)C' instead of (15)N during the t(1) period. Like HN(C)N, these spectra also exhibit special patterns of self and sequential peaks around glycines and prolines, which enable direct identification of certain triplets of residues and thus provide internal checks during the sequential assignment walk. The spectra enable rapid and unambiguous assignment of H(N), (15)N and (13)C(alpha) (or (13)C') in a single experiment, and thus would be of great value in high-throughput structural proteomics. PMID:20643567

  12. 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

  13. Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy.

    PubMed

    Barchi, J J; Grasberger, B; Gronenborn, A M; Clore, G M

    1994-01-01

    The backbone dynamics of the immunoglobulin-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an analysis of the T1/T2 ratios was 3.3 ns at 26 degrees C. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheet arranged in a -1, +3x, -1 topology, on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A number of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (< 20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40 degrees) jumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin.

  15. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin. PMID:21818779

  16. Direct molecular dynamics observation of protein folding transition state ensemble.

    PubMed Central

    Ding, Feng; Dokholyan, Nikolay V; Buldyrev, Sergey V; Stanley, H Eugene; Shakhnovich, Eugene I

    2002-01-01

    The concept of the protein transition state ensemble (TSE), a collection of the conformations that have 50% probability to convert rapidly to the folded state and 50% chance to rapidly unfold, constitutes the basis of the modern interpretation of protein engineering experiments. It has been conjectured that conformations constituting the TSE in many proteins are the expanded and distorted forms of the native state built around a specific folding nucleus. This view has been supported by a number of on-lattice and off-lattice simulations. Here we report a direct observation and characterization of the TSE by molecular dynamic folding simulations of the C-Src SH3 domain, a small protein that has been extensively studied experimentally. Our analysis reveals a set of key interactions between residues, conserved by evolution, that must be formed to enter the kinetic basin of attraction of the native state. PMID:12496119

  17. A polarizable force field for computing the infrared spectra of the polypeptide backbone.

    PubMed

    Schultheis, Verena; Reichold, Rudolf; Schropp, Bernhard; Tavan, Paul

    2008-10-01

    The shapes of the amide bands in the infrared (IR) spectra of proteins and peptides are caused by electrostatically coupled vibrations within the polypeptide backbone and code the structures of these biopolymers. A structural decoding of the amide bands has to resort to simplified models because the huge size of these macromolecules prevents the application of accurate quantum mechanical methods such as density functional theory (DFT). Previous models employed transition-dipole coupling methods that are of limited accuracy. Here we propose a concept for the computation of protein IR spectra, which describes the molecular mechanics (MM) of polypeptide backbones by a polarizable force field of "type II". By extending the concepts of conventional polarizable MM force fields, such a PMM/II approach employs field-dependent parameters not only for the electrostatic signatures of the molecular components but also for the local potentials modeling the stiffness of chemical bonds with respect to elongations, angle deformations, and torsions. Using a PMM/II force field, the IR spectra of the polypeptide backbone can be efficiently calculated from the time dependence of the backbone's dipole moment during a short (e.g., 100 ps) MD simulation by Fourier transformation. PMM/II parameters are derived for harmonic bonding potentials of amide groups in polypeptides from a series of DFT calculations on the model molecule N-methylacetamide (NMA) exposed to homogeneous external electric fields. The amide force constants are shown to vary by as much as 20% for relevant field strengths. As a proof of principle, it is shown that the large solvatochromic effects observed in the IR spectra of NMA upon transfer from the gas phase into aqueous solution are not only excellently reproduced by DFT/MM simulations but are also nicely modeled by the PMM/II approach. The tasks remaining for a proof of practice are specified.

  18. 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 ...

  19. 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

  20. 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.

  1. Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

    PubMed

    Harris, Paul W R; Brimble, Margaret A

    2015-03-01

    The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein.

  2. 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

  3. 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.

  4. The origin of the dynamic transition in proteins

    SciTech Connect

    Khodadadi, S; Pawlus, S; Roh, J H; Garcia Sakai, V; Mamontov, Eugene; Sokolov, A P

    2008-06-01

    Despite extensive efforts in experimental and computational studies, the microscopic understanding of dynamics of biological macromolecules remains a great challenge. It is known that hydrated proteins, DNA and RNA, exhibit a so-called 'dynamic transition'. It appears as a sharp rise of their mean-squared atomic displacements <{sup 2}> at temperatures above 200-230 K. Even after a long history of studies, this sudden activation of biomolecular dynamics remains a puzzle and many contradicting models have been proposed. By combining neutron and dielectric spectroscopy data, we were able to follow protein dynamics over an extremely broad frequency range. Our results show that there is no sudden change in the dynamics of the protein at temperatures around {approx}200-230 K. The protein's relaxation time exhibits a smooth temperature variation over the temperature range of 180-300 K. Thus the experimentally observed sharp rise in is just a result of the protein's structural relaxation reaching the limit of the experimental frequency window. The microscopic mechanism of the protein's structural relaxation remains unclear.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. Disorder transitions and conformational diversity cooperatively modulate biological function in proteins.

    PubMed

    Zea, Diego Javier; Monzon, Alexander Miguel; Gonzalez, Claudia; Fornasari, María Silvina; Tosatto, Silvio C E; Parisi, Gustavo

    2016-06-01

    Structural differences between conformers sustain protein biological function. Here, we studied in a large dataset of 745 intrinsically disordered proteins, how ordered-disordered transitions modulate structural differences between conformers as derived from crystallographic data. We found that almost 50% of the proteins studied show no transitions and have low conformational diversity while the rest show transitions and a higher conformational diversity. In this last subset, 60% of the proteins become more ordered after ligand binding, while 40% more disordered. As protein conformational diversity is inherently connected with protein function our analysis suggests differences in structure-function relationships related to order-disorder transitions.

  10. 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.

  11. Sequence Recognition of DNA by Protein-Induced Conformational Transitions

    SciTech Connect

    Watkins, Derrick; Mohan, Srividya; Koudelka, Gerald B.; Williams, Loren Dean

    2010-11-09

    The binding of proteins to specific sequences of DNA is an important feature of virtually all DNA transactions. Proteins recognize specific DNA sequences using both direct readout (sensing types and positions of DNA functional groups) and indirect readout (sensing DNA conformation and deformability). Previously we showed that the P22 c2 repressor N-terminal domain (P22R NTD) forces the central non-contacted 5{prime}-ATAT-3{prime} sequence of the DNA operator into the B{prime} state, a state known to affect DNA hydration, rigidity and bending. Usually the B{prime} state, with a narrow minor groove and a spine of hydration, is reserved for A-tract DNA (TpA steps disrupt A-tracts). Here, we have co-crystallized P22R NTD with an operator containing a central 5{prime}-ACGT-3{prime} sequence in the non-contacted region. C {center_dot} G base pairs have not previously been observed in the B{prime} state and are thought to prevent it. However, P22R NTD induces a narrow minor groove and a spine of hydration to 5{prime}-ACGT-3{prime}. We observe that C {center_dot} G base pairs have distinctive destabilizing and disordering effects on the spine of hydration. It appears that the reduced stability of the spine results in a higher energy cost for the B to B{prime} transition. The differential effect of DNA sequence on the barrier to this transition allows the protein to sense the non-contacted DNA sequence.

  12. Transitions of protein traffic from cardiac ER to junctional SR

    PubMed Central

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQD-sRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (delTRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and del TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic. PMID:25640161

  13. 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.

  14. 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.

  15. Parathyroid hormone-related protein promotes epithelial-mesenchymal transition.

    PubMed

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

    2010-02-01

    Epithelial-mesenchymal transition (EMT) is an important process that contributes to renal fibrogenesis. TGF-beta1 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-beta and EGF to modulate EMT is unknown. Here, PTHrP(1-36) increased TGF-beta1 in cultured tubuloepithelial cells and TGF-beta blockade inhibited PTHrP-induced EMT-related changes, including upregulation of alpha-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-beta1 and VEGF by PTHrP(1-36). Moreover, inhibition of ERK1/2 blocked EMT induced by either PTHrP(1-36), TGF-beta1, EGF, or VEGF. In vivo, obstruction of mouse kidneys led to changes consistent with EMT and upregulation of TGF-beta1 mRNA, p-EGFR protein, and PTHrP. Taken together, these data suggest that PTHrP, TGF-beta, EGF, and VEGF might cooperate through activation of ERK1/2 to induce EMT in renal tubuloepithelial cells.

  16. 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

  17. 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

  18. 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).

  19. Application of physical organic chemistry to engineered mutants of proteins: Hammond postulate behavior in the transition state of protein folding.

    PubMed Central

    Matouschek, A; Fersht, A R

    1993-01-01

    Transition states in protein folding may be analyzed by linear free-energy relationships (LFERs) analogous to the Brønsted equation for changes in reactivity with changes in structure. There is an additional source of LFERs in protein folding: the perturbation of the equilibrium and rate constants by denaturants. These LFERs give a measure of the position of the transition state along the reaction coordinate. The transition state for folding/unfolding of barnase has been analyzed by both types of LFERs: changing the structure by protein engineering and perturbation by denaturants. The combination has allowed the direct monitoring of Hammond postulate behavior of the transition state on the reaction pathway. Movement of the transition state has been found and analyzed to give further details of the order of events in protein folding. PMID:8356089

  20. 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

  1. Comb-like amphiphilic copolymers bearing acetal-functionalized backbones with the ability of acid-triggered hydrophobic-to-hydrophilic transition as effective nanocarriers for intracellular release of curcumin.

    PubMed

    Zhao, Junqiang; Wang, Haiyang; Liu, Jinjian; Deng, Liandong; Liu, Jianfeng; Dong, Anjie; Zhang, Jianhua

    2013-11-11

    The pH-responsive micelles have enormous potential as nanosized drug carriers for cancer therapy due to their physicochemical changes in response to the tumor intracellular acidic microenvironment. Herein, a series of comb-like amphiphilic copolymers bearing acetal-functionalized backbone were developed based on poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate-co-poly(ethylene glycol) methyl ether methacrylate] [P(TTMA-co-mPEGMA)] as effective nanocarriers for intracellular curcumin (CUR) release. P(TTMA-co-mPEGMA) copolymers with different hydrophobic-hydrophilic ratios were prepared by one-step reversible addition fragmentation chain transfer (RAFT) copolymerization of TTMA and mPEGMA. Their molecular structures and chemical compositions were confirmed by (1)H NMR, Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). P(TTMA-co-mPEGMA) copolymers could self-assemble into nanosized micelles in aqueous solution and displayed low critical micelle concentration (CMC). All P(TTMA-co-mPEGMA) micelles displayed excellent drug loading capacity, due to the strong π-π conjugate action and hydrophobic interaction between the PTTMA and CUR. Moreover, the hydrophobic PTTMA chain could be selectively hydrolyzed into a hydrophilic backbone in the mildly acidic environment, leading to significant swelling and final disassembly of the micelles. These morphological changes of P(TTMA-co-mPEGMA) micelles with time at pH 5.0 were determined by DLS and TEM. The in vitro CUR release from the micelles exhibited a pH-dependent behavior. The release rate of CUR was significantly accelerated at mildly acidic pH of 4.0 and 5.0 compared to that at pH 7.4. Toxicity test revealed that the P(TTMA-co-mPEGMA) copolymers exhibited low cytotoxicity, whereas the CUR-loaded micelles maintained high cytotoxicity for HepG-2 and EC-109 cells. The results indicated that the novel P(TTMA-co-mPEGMA) micelles with low CMC, small and tunable

  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. 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

  4. 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.

  5. Exercise: The Backbone of Spine Treatment

    MedlinePlus

    Exercise: The Backbone of Spine Treatment | View Video Back About Video Struggling with Low Back Pain? Many people are surprised to learn that carefully selected exercise can actually reduce back pain. Some exercises can ...

  6. Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins.

    PubMed

    Ji, Hong-Fang; Chen, Lei; Zhang, Hong-Yu

    2008-08-01

    Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre-protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website.

  7. Error tolerant NMR backbone resonance assignment and automated structure generation.

    PubMed

    Alipanahi, Babak; Gao, Xin; Karakoc, Emre; Li, Shuai Cheng; Balbach, Frank; Feng, Guangyu; Donaldson, Logan; Li, Ming

    2011-02-01

    Error tolerant backbone resonance assignment is the cornerstone of the NMR structure determination process. Although a variety of assignment approaches have been developed, none works sufficiently well on noisy fully automatically picked peaks to enable the subsequent automatic structure determination steps. We have designed an integer linear programming (ILP) based assignment system (IPASS) that has enabled fully automatic protein structure determination for four test proteins. IPASS employs probabilistic spin system typing based on chemical shifts and secondary structure predictions. Furthermore, IPASS extracts connectivity information from the inter-residue information and the (automatically picked) (15)N-edited NOESY peaks which are then used to fix reliable fragments. When applied to automatically picked peaks for real proteins, IPASS achieves an average precision and recall of 82% and 63%, respectively. In contrast, the next best method, MARS, achieves an average precision and recall of 77% and 36%, respectively. The assignments generated by IPASS are then fed into our protein structure calculation system, FALCON-NMR, to determine the 3D structures without human intervention. The final models have backbone RMSDs of 1.25Å, 0.88Å, 1.49Å, and 0.67Å to the reference native structures for proteins TM1112, CASKIN, VRAR, and HACS1, respectively. The web server is publicly available at http://monod.uwaterloo.ca/nmr/ipass.

  8. Understanding traffic dynamics at a backbone POP

    NASA Astrophysics Data System (ADS)

    Taft, Nina; Bhattacharyya, Supratik; Jetcheva, Jorjeta; Diot, Christophe

    2001-07-01

    Spatial and temporal information about traffic dynamics is central to the design of effective traffic engineering practices for IP backbones. In this paper we study backbone traffic dynamics using data collected at a major POP on a tier-1 IP backbone. We develop a methodology that combines packet-level traces from access links in the POP and BGP routing information to build components of POP-to-POP traffic matrices. Our results show that there is wide disparity in the volume of traffic headed towards different egress POPs. At the same time, we find that current routing practices in the backbone tend to constrain traffic between ingress-egress POP pairs to a small number of paths. As a result, there is a wide variation in the utilization level of links in the backbone. Frequent capacity upgrades of the heavily used links are expensive; the need for such upgrades can be reduced by designing load balancing policies that will route more traffic over less utilized links. We identify traffic aggregates based on destination address prefixes and find that this set of criteria isolates a few aggregates that account for an overwhelmingly large portion of inter-POP traffic. We also demonstrate that these aggregates exhibit stability throughout the day on per-hour time scales, and thus they form a natural basis for splitting traffic over multiple paths in order to improve load balancing.

  9. 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.

  10. 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

  11. 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.

  12. Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2.

    PubMed Central

    Li, A; Daggett, V

    1994-01-01

    Temperature-induced unfolding of chymotrypsin inhibitor 2 in water was investigated by molecular dynamics simulations. The major transition state of unfolding was identified on the basis of structural and conformational changes in the protein during the unfolding reaction. The native tertiary contacts in the hydrophobic core were considerably disrupted in the transition state, whereas the secondary structure was partially intact. The extent of structural change of the protein around a particular residue was represented quantitatively by the ratio of the number of contacts the residue makes in the transition state relative to the native state, phi MD, which allows quantitative comparison with the experimentally determined phi F values. For the region of the unfolding trajectory that is identified as the transition state, the phi MD and phi F values are in good agreement, suggesting that the transition state identified in the unfolding simulation corresponds to that probed with protein engineering methods. Although speculative, the transition state identified in the simulation is consistent with available experimental data and provides an in-depth view of what the transition state of unfolding may look like. Images PMID:7937969

  13. 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).

  14. 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.

  15. 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).

  16. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    PubMed

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward.

  17. 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

  18. Dynameomics: A Consensus View of the Protein Unfolding/Folding Transition State Ensemble across a Diverse Set of Protein Folds

    PubMed Central

    Jonsson, Amanda L.; Scott, Kathryn A.; Daggett, Valerie

    2009-01-01

    Abstract The Dynameomics project aims to simulate a representative sample of all globular protein metafolds under both native and unfolding conditions. We have identified protein unfolding transition state (TS) ensembles from multiple molecular dynamics simulations of high-temperature unfolding in 183 structurally distinct proteins. These data can be used to study individual proteins and individual protein metafolds and to mine for TS structural features common across all proteins. Separating the TS structures into four different fold classes (all proteins, all-α, all-β, and mixed α/β and α + β) resulted in no significant difference in the overall protein properties. The residues with the most contacts in the native state lost the most contacts in the TS ensemble. On average, residues beginning in an α-helix maintained more structure in the TS ensemble than did residues starting in β-strands or any other conformation. The metafolds studied here represent 67% of all known protein structures, and this is, to our knowledge, the largest, most comprehensive study of the protein folding/unfolding TS ensemble to date. One might have expected broad distributions in the average global properties of the TS relative to the native state, indicating variability in the amount of structure present in the TS. Instead, the average global properties converged with low standard deviations across metafolds, suggesting that there are general rules governing the structure and properties of the TS. PMID:19948125

  19. 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)

  20. 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

  1. Multi-basin dynamics of a protein in aqueous solution

    SciTech Connect

    Garcia, A.E.

    1994-10-01

    A molecular dynamics simulation of crambin in aqueous solution shows that motions are characteristic of non-linear systems. The authors describe typical non-linear excitations, such as intermittency, for various representations of the protein dynamics and structure. The protein backbone dihedral angles show fast correlated transitions from one minimum well to another. Each transition is followed by small overdamped oscillations. Equal-time cross correlations of all ({phi},{psi}) angles show that correlations are extended along the backbone chain. An analysis based on a generalized least squares fitting of the protein fluctuations along vectors show that a small set of molecule optimal dynamic coordinates (MODC) describe most of the protein fluctuations. In addition, the MODC describe a trajectory where the protein conformation jumps from one minimum well to another. An extension of the MODC describing 2- and 3-dimensional cuts of the protein configurational space clearly shows a trajectory around multiple basins of attraction.

  2. Predicting order of conformational changes during protein conformational transitions using an interpolated elastic network model.

    PubMed

    Tekpinar, Mustafa; Zheng, Wenjun

    2010-08-15

    The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions of various biological nanomachines. Coarse-grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse-grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP-driven transitions in three important biological nanomachines (myosin, F(1) ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines.

  3. 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

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

    PubMed

    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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. Modulation of a cytoskeletal calpain-like protein induces major transitions in trypanosome morphology.

    PubMed

    Hayes, Polly; Varga, Vladimir; Olego-Fernandez, Sofia; Sunter, Jack; Ginger, Michael L; Gull, Keith

    2014-08-01

    Individual eukaryotic microbes, such as the kinetoplastid parasite Trypanosoma brucei, have a defined size, shape, and form yet transition through life cycle stages, each having a distinct morphology. In questioning the structural processes involved in these transitions, we have identified a large calpain-like protein that contains numerous GM6 repeats (ClpGM6) involved in determining T. brucei cell shape, size, and form. ClpGM6 is a cytoskeletal protein located within the flagellum along the flagellar attachment zone (FAZ). Depletion of ClpGM6 in trypomastigote forms produces cells with long free flagella and a shorter FAZ, accompanied by repositioning of the basal body, the kinetoplast, Golgi, and flagellar pocket, reflecting an epimastigote-like morphology. Hence, major changes in microbial cell form can be achieved by simple modulation of one or a few proteins via coordinated association and positioning of membrane and cytoskeletal components.

  10. Coil–globule transition in the denatured state of a small protein

    PubMed Central

    Sherman, Eilon; Haran, Gilad

    2006-01-01

    Upon transfer from strongly denaturing to native conditions, proteins undergo a collapse that either precedes folding or occurs simultaneously with it. This collapse is similar to the well known coil–globule transition of polymers. Here we employ single-molecule fluorescence methods to fully characterize the equilibrium coil–globule transition in the denatured state of the IgG-binding domain of protein L. By using FRET measurements on freely diffusing individual molecules, we determine the radius of gyration of the protein, which shows a gradual expansion as the concentration of the denaturant, guanidinium hydrochloride, is increased all the way up to 7 M. This expansion is observed also in fluorescence correlation spectroscopy measurements of the hydrodynamic radius of the protein. We analyze the radius of gyration measurements using the theory of the coil–globule transition of Sanchez [Sanchez, I. C. (1979) Macromolecules 12, 980–988], which balances the excluded volume entropy of the chain with the average interresidue interaction energy. In particular, we calculate the solvation energy of the denatured protein, a property that is not readily accessible in other experiments. The dependence of this energy on denaturant concentration is nonlinear, contrasting with the common linear extrapolation method used to describe denaturation energy. Interestingly, a fit to the binding model of chemical denaturation suggests a single denaturant binding site per protein residue. The size of the denatured protein under native conditions can be extrapolated from the data as well, showing that the fully collapsed state of protein is only ≈10% larger than the folded state. PMID:16857738

  11. Telephone wire is backbone of security system

    SciTech Connect

    Brede, K.; Rackson, L.T.

    1995-09-01

    Video provides a variety of low-cost, high-quality solutions in today`s security environment. Cost-conscious managers of power generation stations, casinos, prison facilities, military bases and office buildings are considering using regular telephone wire (unshielded twisted pair-UTP) within their existing systems as the backbone of a video to the PC, personal and video-conferencing and training are other areas where phone wire in a building can save money and provide an alternative to coax or fiber for video. More and more, businesses and government agencies are meeting their needs efficiently by using telephone wires for more than just telephones.

  12. Globular-disorder transition in proteins: a compromise between hydrophobic and electrostatic interactions?

    PubMed

    Baruah, Anupaul; Biswas, Parbati

    2016-08-17

    The charge-hydrophobicity correlation of globular and disordered proteins is explored using a generalized self-consistent field theoretical method combined with Monte Carlo simulations. Globular and disordered protein sequences with varied mean net charge and mean hydrophobicity are designed by theory, while Metropolis Monte Carlo generates a suitable ensemble of conformations. Results imply a transition of the dominant interactions between globular and disordered proteins across the charge-hydrophobicity boundary. It is observed that the charge-hydrophobicity boundary actually represents a trade-off between the repulsive and attractive interactions in a protein sequence. The attractive interactions predominate on the globular side of the boundary, while the repulsive interactions prevail on the disordered side. For globular proteins, core forming hydrophobic interactions are dominant leading to a minimally frustrated native conformation. For disordered proteins, the repulsive electrostatic interactions prevail yielding a minimally frustrated region comprising of an expanded, dynamic conformational ensemble. Thus, protein disorder, like protein folding, satisfies the principle of minimal frustration. All results are compared to real globular and disordered proteins. Thus this algorithm may be useful to probe the conformational characteristics of disordered proteins. PMID:27498593

  13. Backbone and side-chain ¹H, ¹³C and ¹⁵N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii.

    PubMed

    Tse, Man-Kit; Cheung, Stanley K K; Ke, Yi-hong; Lau, Candy C Y; Sze, Kong-Hung; Yuen, Kwok-Yung

    2014-04-01

    Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.

  14. Determination of the glass-transition temperature of proteins from a viscometric approach.

    PubMed

    Monkos, Karol

    2015-03-01

    All fully hydrated proteins undergo a distinct change in their dynamical properties at glass-transition temperature Tg. To determine indirectly this temperature for dry albumins, the viscosity measurements of aqueous solutions of human, equine, ovine, porcine and rabbit serum albumin have been conducted at a wide range of concentrations and at temperatures ranging from 278 K to 318 K. Viscosity-temperature dependence of the solutions is discussed on the basis of the three parameters equation resulting from Avramov's model. One of the parameter in the Avramov's equation is the glass-transition temperature. For all studied albumins, Tg of a solution monotonically increases with increasing concentration. The glass-transition temperature of a solution depends both on Tg for a dissolved dry protein Tg,p and water Tg,w. To obtain Tg,p for each studied albumin the modified Gordon-Taylor equation was applied. This equation describes the dependence of Tg of a solution on concentration, and Tg,p and a parameter depending on the strength of the protein-solvent interaction are the fitting parameters. Thus determined the glass-transition temperature for the studied dry albumins is in the range (215.4-245.5)K.

  15. Self-similarity of biopolymer backbones in the ribosome

    NASA Astrophysics Data System (ADS)

    Lee, Chang-Yong

    2008-08-01

    Self-similar properties of the biopolymer backbones in the ribosome are investigated in terms of the fractal dimension. We especially estimate the chain fractal and capacity dimensions of the ribosomal RNAs and proteins, which are constituents of the ribosome. The fractal dimensions of both biopolymers are compared with that of the self-avoiding walk, which is a typical model of a polymer without interaction between monomers. We demonstrate that the fractality found in the ribosomal RNAs is pertinent to explain their structural characteristics: local helix formation and long-range tertiary interaction forming three-dimensional structures. The fractal dimension of the ribosomal protein supports the existence of the long and extended domain, which is hardly seen in the globular protein. The self-similarity also upholds the fact that the ribosomal proteins function primarily to stabilize the structure of the ribosome by both the long-extended domain of the protein penetrating into the inside of the RNA, and the globular domain interacting with the RNA on the exterior of it. These results partially, if not whole, unravel the structural characteristics of the biopolymers in the ribosome.

  16. 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

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

    DOE PAGES

    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

  18. 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.

  19. 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

  20. Transits

    NASA Astrophysics Data System (ADS)

    Gilliland, Ronald L.

    Transits of the planets Mercury and especially Venus have been exciting events in the development of astronomy over the past few hundred years. Just two years ago the first transiting extra-solar planet, HD 209458b, was discovered, and subsequent studies during transit have contributed fundamental new knowledge. From the photometric light curve during transit one obtains a basic confirmation that the radial velocity detected object is indeed a planet by allowing precise determination of its mass and radius relative to these stellar quantities. From study of spectroscopic changes during transit it has been possible to probe for individual components of the transiting planets atmosphere. Planet transits are likely to become a primary tool for detection of new planets, especially other Earth-like planets with the Kepler Discovery Mission. Looking ahead, the additional aperture of the James Webb Space Space Telescope promises to allow the first possibility of studying the atmosphere of extra-solar Earth-analogue planets, perhaps even providing the first evidence of direct relevance to the search for signs of life on other planets.

  1. Engineering the polymer backbone to strengthen nonfouling sulfobetaine hydrogels.

    PubMed

    Carr, Louisa; Cheng, Gang; Xue, Hong; Jiang, Shaoyi

    2010-09-21

    We have demonstrated that molecularly engineering the chemical structure of a monomer can lead to hydrogels with improved mechanical strength. In this case, hydrogels from zwitterionic sulfobetaine methacrylate monomers were compared to sulfobetaine vinylimidazole (pSBVI) hydrogels. We show that the introduction of the vinylimidazole backbone improves the tensile and compressive mechanical properties of the sulfobetaine hydrogel by an order of magnitude over the same properties of a methacrylate hydrogel. Zwitterionic groups have been shown to create surface coating materials with ultralow fouling properties, and we demonstrate here that the presence of the imidazole group does not compromise the nonfouling properties attributed to the zwitterionic sulfobetaine: surfaces coated with pSBVI exhibited exceptionally low nonspecific protein adsorption, and cell adhesion was reduced by 97% relative to low-fouling poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels. PMID:20731337

  2. NMR study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

    PubMed

    Melekis, Efstathios; Tsika, Aikaterini C; Lichière, Julie; Chasapis, Christos T; Margiolaki, Irene; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-04-01

    Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

  3. 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.

  4. 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.

  5. Transition metal complexes as mediator-titrants in protein redox potentiometry.

    PubMed

    Bernhardt, Paul V; Chen, Kuan-I; Sharpe, Philip C

    2006-10-01

    A selection of nine macrocyclic Fe(III/II) and Co(III/II) transition metal complexes has been chosen to serve as a universal set of mediator-titrants in redox potentiometry of protein samples. The potential range spanned by these mediators is approximately from +300 to -700 mV vs the normal hydrogen electrode, which covers the range of most protein redox potentials accessible in aqueous solution. The complexes employed exhibit stability in both their oxidized and their reduced forms as well as pH-independent redox potentials within the range 6 < pH < 9. The mediators were also chosen on the basis of their very weak visible absorption maxima in both oxidation states, which will enable (for the first time) optical redox potentiometric titrations of proteins with relatively low extinction coefficients. This has previously been impractical with organic mediators, such as indoles, viologens and quinones, whose optical spectra interfere strongly with those of the protein.

  6. Conformational transition paths harbor structures useful for aiding drug discovery and understanding enzymatic mechanisms in protein kinases.

    PubMed

    Wong, Chung F

    2016-01-01

    This short article examines the usefulness of fast simulations of conformational transition paths in elucidating enzymatic mechanisms and guiding drug discovery for protein kinases. It applies the transition path method in the MOIL software package to simulate the paths of conformational transitions between six pairs of structures from the Protein Data Bank. The structures along the transition paths were found to resemble experimental structures that mimic transient structures believed to form during enzymatic catalysis or conformational transitions, or structures that have drug candidates bound. These findings suggest that such simulations could provide quick initial insights into the enzymatic mechanisms or pathways of conformational transitions of proteins kinases, or could provide structures useful for aiding structure-based drug design.

  7. Predicting folding-unfolding transitions in proteins without a priori knowledge of the folded state

    NASA Astrophysics Data System (ADS)

    Okan, Osman; Turgut, Deniz; Garcia, Angel; Ozisik, Rahmi

    2013-03-01

    The common computational method of studying folding transitions in proteins is to compare simulated conformations against the folded structure, but this method obviously requires the folded structure to be known beforehand. In the current study, we show that the use of bond orientational order parameter (BOOP) Ql [Steinhardt PJ, Nelson DR, Ronchetti M, Phys. Rev. B 1983, 28, 784] is a viable alternative to the commonly adopted root mean squared distance (RMSD) measure in probing conformational transitions. Replica exchange molecular dynamics simulations of the trp-cage protein (with 20 residues) in TIP-3P water were used to compare BOOP against RMSD. The results indicate that the correspondence between BOOP and RMSD time series become stronger with increasing l. We finally show that robust linear models that incorporate different Ql can be parameterized from a given replica run and can be used to study other replica trajectories. This work is partially supported by NSF DUE-1003574.

  8. 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.

  9. Sofosbuvir as backbone of interferon free treatments.

    PubMed

    Bourlière, Marc; Oules, Valèrie; Ansaldi, Christelle; Adhoute, Xavier; Castellani, Paul

    2014-12-15

    Sofosbuvir is the first-in-class NS5B nucleotide analogues to be launched for hepatitis C virus (HCV) treatment. Its viral potency, pangenotypic activity and high barrier to resistance make it the ideal candidate to become a backbone for several IFN-free regimens. Recent data demonstrated that sofosbuvir either with ribavirin alone or in combination with other direct-acting antivirals (DAAs) as daclatasvir, ledipasvir or simeprevir are able to cure HCV in at least 90% or over of patients. Treatment experienced genotype 3 population may remain the most difficult to treat population, but ongoing DAA combination studies will help to fill this gap. Safety profile of sofosbuvir or combination with other DAAs is good. Resistance to sofosbuvir did not appear as a significant issue. The rationale for using this class of drug and the available clinical data are reviewed.

  10. 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.

  11. 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

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

    2014-04-01

    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 Escherichia 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 (1)H, (13)C, and (15)N 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.

  12. Thiol redox transitions by thioredoxin and thioredoxin-binding protein-2 in cell signaling.

    PubMed

    Yoshihara, Eiji; Chen, Zhe; Matsuo, Yoshiyuki; Masutani, Hiroshi; Yodoi, Junji

    2010-01-01

    The cellular thiol redox state is a crucial mediator of metabolic, signaling and transcriptional processes in cells, and an exquisite balance between the oxidizing and reducing states is essential for the normal function and survival of cells. Reactive oxygen species (ROS) are widely known to function as a kind of second messenger for intracellular signaling and to modulate the thiol redox state. Thiol reduction is mainly controlled by the thioredoxin (TRX) system and glutathione (GSH) systems as scavengers of ROS and regulators of the protein redox states. The thioredoxin system is composed of several related molecules interacting through the cysteine residues at the active site, including thioredoxin, thioredoxin-2, a mitochondrial thioredoxin family, and transmembrane thioredoxin-related protein (TMX), an endoplasmic reticulum (ER)-specific thioredoxin family. Thioredoxin couples with thioredoxin-dependent peroxidases (peroxiredoxin) to scavenge hydrogen peroxide. In addition, thioredoxin does not simply act only as a scavenger of ROS but also as an important regulator of oxidative stress response through protein-protein interaction. The interaction of thioredoxin and thioredoxin-binding proteins such as thioredoxin-binding protein-2 (TBP-2, also called as Txnip or VDUP1), apoptosis signal kinase (ASK-1), redox factor 1 (Ref-1), Forkhead box class O 4 (FoxO4), and nod-like receptor proteins (NLRPs) suggested unconventional functions of thioredoxin and a novel mechanism of redox regulation. Here, we introduce the central mechanism of thiol redox transition in cell signaling regulated by thioredoxin and related molecules.

  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.

  14. 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

  15. Configuration-dependent diffusion can shift the kinetic transition state and barrier height of protein folding

    PubMed Central

    Chahine, Jorge; Oliveira, Ronaldo J.; Leite, Vitor B. P.; Wang, Jin

    2007-01-01

    We show that diffusion can play an important role in protein-folding kinetics. We explicitly calculate the diffusion coefficient of protein folding in a lattice model. We found that diffusion typically is configuration- or reaction coordinate-dependent. The diffusion coefficient is found to be decreasing with respect to the progression of folding toward the native state, which is caused by the collapse to a compact state constraining the configurational space for exploration. The configuration- or position-dependent diffusion coefficient has a significant contribution to the kinetics in addition to the thermodynamic free-energy barrier. It effectively changes (increases in this case) the kinetic barrier height as well as the position of the corresponding transition state and therefore modifies the folding kinetic rates as well as the kinetic routes. The resulting folding time, by considering both kinetic diffusion and the thermodynamic folding free-energy profile, thus is slower than the estimation from the thermodynamic free-energy barrier with constant diffusion but is consistent with the results from kinetic simulations. The configuration- or coordinate-dependent diffusion is especially important with respect to fast folding, when there is a small or no free-energy barrier and kinetics is controlled by diffusion. Including the configurational dependence will challenge the transition state theory of protein folding. The classical transition state theory will have to be modified to be consistent. The more detailed folding mechanistic studies involving phi value analysis based on the classical transition state theory also will have to be modified quantitatively. PMID:17804812

  16. NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

    PubMed

    Makrynitsa, Garyfallia I; Ntonti, Dioni; Marousis, Konstantinos D; Tsika, Aikaterini C; Lichière, Julie; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-10-01

    Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

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

    PubMed Central

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

    2016-01-01

    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-nic operon, 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 trans negatively interferes with mini-RA3 replication and stable maintenance of RA3. PMID:26850301

  18. 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

  19. Phase Transitions of Spindle-Associated Protein Regulate Spindle Apparatus Assembly

    PubMed Central

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

    2015-01-01

    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

  20. Mapping the Structure and Conformational Movements of Proteins with Transition Metal Ion FRET

    SciTech Connect

    Taraska, J.; Puljung, M; Olivier, N; Olivier, G; Zagotta, W

    2009-01-01

    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 fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine 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. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.

  1. 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

  2. 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

  3. 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

  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. Melatonin mediates the stabilization of DELLA proteins to repress the floral transition in Arabidopsis.

    PubMed

    Shi, Haitao; Wei, Yunxie; Wang, Qiannan; Reiter, Russel J; He, Chaozu

    2016-04-01

    Precise floral transition from vegetative growth phase to reproductive growth phase is very important in plant life cycle. In flowering genetic pathways, DELLA proteins are master transcriptional regulators of gibberelic acid (GA) pathway, and FLOWERING LOCUS C (FLC) is a core repressor of vernalization pathway as well as downstream of DELLAs. As a crucial messenger in plants, the possible involvement of melatonin (N-acetyl-5-methoxytryptamine) in flowering and underlying molecular mechanism are unknown in Arabidopsis. In this study, we found that exogenous melatonin treatment delayed floral transition in Arabidopsis. Exogenous melatonin treatment conferred protein stabilizations of DELLAs [REPRESSOR of ga1-3 (RGA) and RGA-LIKE3 (RGL3)], without regulating the transcripts of DELLAs and endogenous GA level. Notably, exogenous melatonin delayed plant flowering and DELLA-activated transcripts of FLC were alleviated in della mutants, and those were exacerbated in DELLA overexpressing plants. Taken together, this study provides direct link between melatonin and floral transition, and indicates the novel involvement of DELLAs-activated FLC in melatonin-mediated flowering in Arabidopsis.

  6. 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

  7. Tuning backbones and side-chains of cationic conjugated polymers for optical signal amplification of fluorescent DNA detection.

    PubMed

    Huang, Yan-Qin; Liu, Xing-Fen; Fan, Qu-Li; Wang, Lihua; Song, Shiping; Wang, Lian-Hui; Fan, Chunhai; Huang, Wei

    2009-06-15

    Three cationic conjugated polymers (CCPs) exhibiting different backbone geometries and charge densities were used to investigate how their conjugated backbone and side chain properties, together with the transitions of DNA amphiphilic properties, interplay in the CCP/DNA-C* (DNA-C*: fluorophore-labeled DNA) complexes to influence the optical signal amplification of fluorescent DNA detection based on Förster resonance energy transfer (FRET). By examining the FRET efficiencies to dsDNA-C* (dsDNA: double-stranded DNA) and ssDNA-C* (ssDNA: single-stranded DNA) for each CCP, twisted conjugated backbones and higher charge densities were proved to facilitate electrostatic attraction in CCP/dsDNA-C* complexes, and induced improved sensitivity to DNA hybridization. Especially, by using the CCP with twisted conjugated backbone and the highest charge density, a more than 7-fold higher efficiency of FRET to dsDNA-C* was found than to ssDNA-C*, indicating a high signal amplification for discriminating between dsDNA and ssDNA. By contrast, linear conjugated backbones and lower charge density were demonstrated to favor hydrophobic interactions in CCP/ssDNA-C* complexes. These findings provided guidelines for the design of novel sensitive CCP, which can be useful to recognize many other important DNA activities involving transitions of DNA amphiphilic properties like DNA hybridization, such as specific DNA binding with ions, some secondary or tertiary structural changes of DNA, and so forth.

  8. Iterative cluster-NMA (icNMA): A tool for generating conformational transitions in proteins

    PubMed Central

    Schuyler, Adam D.; Jernigan, Robert L.; Qasba, Pradman K.; Ramakrishnan, Boopathy; Chirikjian, Gregory S.

    2010-01-01

    Computational models provide insight into the structure-function relationship in proteins. These approaches, especially those based on normal mode analysis, can identify the accessible motion space around a given equilibrium structure. The large magnitude, collective motions identified by these methods are often well aligned with the general direction of the expected conformational transitions. However, these motions cannot realistically be extrapolated beyond the local neighborhood of the starting conformation. In this paper, the icNMA method is presented for traversing the energy landscape from a starting conformation to a desired goal conformation. This is accomplished by allowing the evolving geometry of the intermediate structures to define the local accessible motion space, and thus produce an appropriate displacement. Following the derivation of the icNMA method, a set of sample simulations are performed to probe the robustness of the model. A detailed analysis of β1,4-galactosyltransferase-T1 is also given, to highlight many of the capabilities of icNMA. Remarkably, during the transition, a helix is seen to be extended by an additional turn, emphasizing a new unknown role for secondary structures to absorb slack during transitions. The transition pathway for adenylate kinase, which has been frequently studied in the literature, is also discussed. PMID:18712827

  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. 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

  11. 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

  12. NHC Backbone Configuration in Ruthenium-Catalyzed Olefin Metathesis.

    PubMed

    Paradiso, Veronica; Costabile, Chiara; Grisi, Fabia

    2016-01-20

    The catalytic properties of olefin metathesis ruthenium complexes bearing N-heterocyclic carbene ligands with stereogenic centers on the backbone are described. Differences in catalytic behavior depending on the backbone configurations of symmetrical and unsymmetrical NHCs are discussed. In addition, an overview on asymmetric olefin metathesis promoted by chiral catalysts bearing C₂-symmetric and C₁-symmetric NHCs is provided.

  13. NHC Backbone Configuration in Ruthenium-Catalyzed Olefin Metathesis.

    PubMed

    Paradiso, Veronica; Costabile, Chiara; Grisi, Fabia

    2016-01-01

    The catalytic properties of olefin metathesis ruthenium complexes bearing N-heterocyclic carbene ligands with stereogenic centers on the backbone are described. Differences in catalytic behavior depending on the backbone configurations of symmetrical and unsymmetrical NHCs are discussed. In addition, an overview on asymmetric olefin metathesis promoted by chiral catalysts bearing C₂-symmetric and C₁-symmetric NHCs is provided. PMID:26805793

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Altered Specificity of DNA-Binding Proteins with Transition Metal Dimerization Domains

    NASA Astrophysics Data System (ADS)

    Cuenoud, Bernard; Schepartz, Alanna

    1993-01-01

    The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA. A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides. Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation. These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding. This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

  20. Frequent expression of zinc-finger protein ZNF165 in human urinary bladder transitional cell carcinoma.

    PubMed

    Singh, Pankaj Kumar; Srivastava, Anupam Kumar; Dalela, Divakar; Rath, Srikanta Kumar; Goel, Madhu Mati; Bhatt, Madan Lal Brahma

    2015-01-01

    The aim of the study is to evaluate mRNA/protein expression of zinc finger protein 165 (ZNF165) in transitional cell carcinomas (TCCs) of urinary bladder and correlate its expression with the clinicopathological characteristics of patients. In this study, the methods of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were utilized to evaluate mRNA/protein expression of ZNF165 in TCC. Independent Student's t test, ANOVA and Chi-square (χ(2)) were used to analyze the data statistically. We observed overexpression of ZNF165 mRNA in testis and majority (59.2%) of TCC patients. ZNF165 mRNA expression was also detected in adjacent noncancerous tissues (ANCTs) and some other normal tissues. Relative mean fold expression of ZNF165 mRNA was found to be significantly (p<0.01) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients. (12.11±9.57 vs. 5.72±2.61, p=0.009). ZNF165 protein expression was demonstrated on archival formalin-fixed, paraffin-embedded (FFPE) bladder tissues using IHC and nuclear staining pattern was detected. No significant difference was observed in protein expression of ZNF165 between the two groups (NMIBC and MIBC patients) (61.1% vs. 55.2%, p=0.629). No significant protein expression of ZNF165 was observed among ANCTs and benign prostatic hyperplasia (BPH) used as control. Our study results suggest that ZNF165 mRNA/protein expression was observed in TCC of human urinary bladder and might be used as a novel diagnostic biomarker and as well a vaccine target in development of urinary bladder cancer specific immunotherapy.

  1. How Sensitive is the Amide I Vibration of the Polypeptide Backbone to Electric Fields?

    PubMed

    Oh, Kwang-Im; Fiorin, Giacomo; Gai, Feng

    2015-12-01

    Site-selective isotopic labeling of amide carbonyls offers a nonperturbative means to introduce a localized infrared probe into proteins. Although this strategy has been widely used to investigate various biological questions, the dependence of the underlying amide I vibrational frequency on electric fields (or Stark tuning rate) has not been fully determined, which prevents it from being used in a quantitative manner in certain applications. Herein, through the use of experiments and molecular dynamics simulations, the Stark tuning rate of the amide I vibration of an isotopically labeled backbone carbonyl in a transmembrane α-helix is determined to be approximately 1.4 cm(-1) /(MV/cm). This result provides a quantitative basis for using this vibrational model to assess local electric fields in proteins, among other applications. For instance, by using this value, we are able to show that the backbone region of a dipeptide has a surprisingly low dielectric constant.

  2. Dynamical transition, hydrophobic interface, and the temperature dependence of electrostatic fluctuations in proteins

    NASA Astrophysics Data System (ADS)

    Lebard, David N.; Matyushov, Dmitry V.

    2008-12-01

    Molecular dynamics simulations have revealed a dramatic increase, with increasing temperature, of the amplitude of electrostatic fluctuations caused by water at the active site of metalloprotein plastocyanin. The increased breadth of electrostatic fluctuations, expressed in terms of the reorganization energy of changing the redox state of the protein, is related to the formation of the hydrophobic protein-water interface, allowing large-amplitude collective fluctuations of the water density in the protein’s first solvation shell. On top of the monotonic increase of the reorganization energy with increasing temperature, we have observed a spike at ≃220K also accompanied by a significant slowing of the exponential collective Stokes shift dynamics. In contrast to the local density fluctuations of the hydration-shell waters, these spikes might be related to the global property of the water solvent crossing the Widom line or undergoing a weak first-order transition.

  3. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  4. 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

  5. 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.

  6. 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.

  7. 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.}

  8. 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.

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

    PubMed Central

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

    2015-01-01

    Abstract 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

  10. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  11. 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.

  12. Temperature dependence of fast carbonyl backbone dynamics in chicken villin headpiece subdomain.

    PubMed

    Vugmeyster, Liliya; Ostrovsky, Dmitry

    2011-06-01

    Temperature-dependence of protein dynamics can provide information on details of the free energy landscape by probing the characteristics of the potential responsible for the fluctuations. We have investigated the temperature-dependence of picosecond to nanosecond backbone dynamics at carbonyl carbon sites in chicken villin headpiece subdomain protein using a combination of three NMR relaxation rates: (13)C' longitudinal rate, and two cross-correlated rates involving dipolar and chemical shift anisotropy (CSA) relaxation mechanisms, (13)C'/(13)C'-(13)C(α) CSA/dipolar and (13)C'/(13)C'-(15)N CSA/dipolar. Order parameters have been extracted using the Lipari-Szabo model-free approach assuming a separation of the time scales of internal and molecular motions in the 2-16°C temperature range. There is a gradual deviation from this assumption from lower to higher temperatures, such that above 16°C the separation of the time scales is inconsistent with the experimental data and, thus, the Lipari-Szabo formalism can not be applied. While there are variations among the residues, on the average the order parameters indicate a markedly steeper temperature dependence at backbone carbonyl carbons compared to that probed at amide nitrogens in an earlier study. This strongly advocates for probing sites other than amide nitrogen for accurate characterization of the potential and other thermodynamics characteristics of protein backbone.

  13. Evaluating transition state structures of vanadium-phosphatase protein complexes using shape analysis.

    PubMed

    Sánchez-Lombardo, Irma; Alvarez, Santiago; McLauchlan, Craig C; Crans, Debbie C

    2015-06-01

    Shape analysis of coordination complexes is well-suited to evaluate the subtle distortions in the trigonal bipyramidal (TBPY-5) geometry of vanadium coordinated in the active site of phosphatases and characterized by X-ray crystallography. Recent studies using the tau (τ) analysis support the assertion that vanadium is best described as a trigonal bipyramid, because this geometry is the ideal transition state geometry of the phosphate ester substrate hydrolysis (C.C. McLauchlan, B.J. Peters, G.R. Willsky, D.C. Crans, Coord. Chem. Rev. http://dx.doi.org/10.1016/j.ccr.2014.12.012 ; D.C. Crans, M.L. Tarlton, C.C. McLauchlan, Eur. J. Inorg. Chem. 2014, 4450-4468). Here we use continuous shape measures (CShM) analysis to investigate the structural space of the five-coordinate vanadium-phosphatase complexes associated with mechanistic transformations between the tetrahedral geometry and the five-coordinate high energy TBPY-5 geometry was discussed focusing on the protein tyrosine phosphatase 1B (PTP1B) enzyme. No evidence for square pyramidal geometries was observed in any vanadium-protein complexes. The shape analysis positioned the metal ion and the ligands in the active site reflecting the mechanism of the cleavage of the organic phosphate in a phosphatase. We identified the umbrella distortions to be directly on the reaction path between tetrahedral phosphate and the TBPY-5-types of high-energy species. The umbrella distortions of the trigonal bipyramid are therefore identified as being the most relevant types of transition state structures for the phosphoryl group transfer reactions for phosphatases and this may be related to the possibility that vanadium is an inhibitor for enzymes that support both exploded and five-coordinate transition states.

  14. Constrained proper sampling of conformations of transition state ensemble of protein folding.

    PubMed

    Lin, Ming; Zhang, Jian; Lu, Hsiao-Mei; Chen, Rong; Liang, Jie

    2011-02-21

    Characterizing the conformations of protein in the transition state ensemble (TSE) is important for studying protein folding. A promising approach pioneered by Vendruscolo et al. [Nature (London) 409, 641 (2001)] to study TSE is to generate conformations that satisfy all constraints imposed by the experimentally measured φ values that provide information about the native likeness of the transition states. Faísca et al. [J. Chem. Phys. 129, 095108 (2008)] generated conformations of TSE based on the criterion that, starting from a TS conformation, the probabilities of folding and unfolding are about equal through Markov Chain Monte Carlo (MCMC) simulations. In this study, we use the technique of constrained sequential Monte Carlo method [Lin et al., J. Chem. Phys. 129, 094101 (2008); Zhang et al. Proteins 66, 61 (2007)] to generate TSE conformations of acylphosphatase of 98 residues that satisfy the φ-value constraints, as well as the criterion that each conformation has a folding probability of 0.5 by Monte Carlo simulations. We adopt a two stage process and first generate 5000 contact maps satisfying the φ-value constraints. Each contact map is then used to generate 1000 properly weighted conformations. After clustering similar conformations, we obtain a set of properly weighted samples of 4185 candidate clusters. Representative conformation of each of these cluster is then selected and 50 runs of Markov chain Monte Carlo (MCMC) simulation are carried using a regrowth move set. We then select a subset of 1501 conformations that have equal probabilities to fold and to unfold as the set of TSE. These 1501 samples characterize well the distribution of transition state ensemble conformations of acylphosphatase. Compared with previous studies, our approach can access much wider conformational space and can objectively generate conformations that satisfy the φ-value constraints and the criterion of 0.5 folding probability without bias. In contrast to previous

  15. 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

  16. A backbone lever-arm effect enhances polymer mechanochemistry.

    PubMed

    Klukovich, Hope M; Kouznetsova, Tatiana B; Kean, Zachary S; Lenhardt, Jeremy M; Craig, Stephen L

    2013-02-01

    Mechanical forces along a polymer backbone can be used to bring about remarkable reactivity in embedded mechanically active functional groups, but little attention has been paid to how a given polymer backbone delivers that force to the reactant. Here, single-molecule force spectroscopy was used to directly quantify and compare the forces associated with the ring opening of gem-dibromo and gem-dichlorocyclopropanes affixed along the backbone of cis-polynorbornene and cis-polybutadiene. The critical force for isomerization drops by about one-third in the polynorbornene scaffold relative to polybutadiene. The root of the effect lies in more efficient chemomechanical coupling through the polynorbornene backbone, which acts as a phenomenological lever with greater mechanical advantage than polybutadiene. The experimental results are supported computationally and provide the foundation for a new strategy by which to engineer mechanochemical reactivity. PMID:23344431

  17. A backbone lever-arm effect enhances polymer mechanochemistry

    NASA Astrophysics Data System (ADS)

    Klukovich, Hope M.; Kouznetsova, Tatiana B.; Kean, Zachary S.; Lenhardt, Jeremy M.; Craig, Stephen L.

    2013-02-01

    Mechanical forces along a polymer backbone can be used to bring about remarkable reactivity in embedded mechanically active functional groups, but little attention has been paid to how a given polymer backbone delivers that force to the reactant. Here, single-molecule force spectroscopy was used to directly quantify and compare the forces associated with the ring opening of gem-dibromo and gem-dichlorocyclopropanes affixed along the backbone of cis-polynorbornene and cis-polybutadiene. The critical force for isomerization drops by about one-third in the polynorbornene scaffold relative to polybutadiene. The root of the effect lies in more efficient chemomechanical coupling through the polynorbornene backbone, which acts as a phenomenological lever with greater mechanical advantage than polybutadiene. The experimental results are supported computationally and provide the foundation for a new strategy by which to engineer mechanochemical reactivity.

  18. Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase.

    PubMed

    Xiaoxia, Liu; Marston, James P; Baxter, Nicola J; Hounslow, Andrea M; Yufen, Zhao; Blackburn, G Michael; Cliff, Matthew J; Waltho, Jonathan P

    2011-03-23

    The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion. PMID:21348513

  19. 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

  20. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  2. 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

  3. Radical-driven peptide backbone dissociation tandem mass spectrometry.

    PubMed

    Oh, Han Bin; Moon, Bongjin

    2015-01-01

    In recent years, a number of novel tandem mass spectrometry approaches utilizing radical-driven peptide gas-phase fragmentation chemistry have been developed. These approaches show a peptide fragmentation pattern quite different from that of collision-induced dissociation (CID). The peptide fragmentation features of these approaches share some in common with electron capture dissociation (ECD) or electron transfer dissociation (ETD) without the use of sophisticated equipment such as a Fourier-transform mass spectrometer. For example, Siu and coworkers showed that CID of transition metal (ligand)-peptide ternary complexes led to the formation of peptide radical ions through dissociative electron transfer (Chu et al., 2000. J Phys Chem B 104:3393-3397). The subsequent collisional activation of the generated radical ions resulted in a number of characteristic product ions, including a, c, x, z-type fragments and notable side-chain losses. Another example is the free radical initiated peptide sequencing (FRIPS) approach, in which Porter et al. and Beauchamp et al. independently introduced a free radical initiator to the primary amine group of the lysine side chain or N-terminus of peptides (Masterson et al., 2004. J Am Chem Soc 126:720-721; Hodyss et al., 2005 J Am Chem Soc 127: 12436-12437). Photodetachment of gaseous multiply charged peptide anions (Joly et al., 2008. J Am Chem Soc 130:13832-13833) and UV photodissociation of photolabile radical precursors including a C-I bond (Ly & Julian, 2008. J Am Chem Soc 130:351-358; Ly & Julian, 2009. J Am Soc Mass Spectrom 20:1148-1158) also provide another route to generate radical ions. In this review, we provide a brief summary of recent results obtained through the radical-driven peptide backbone dissociation tandem mass spectrometry approach.

  4. 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

  5. The protein tyrosine phosphatase Pez regulates TGFbeta, epithelial-mesenchymal transition, and organ development.

    PubMed

    Wyatt, Leila; Wadham, Carol; Crocker, Lesley A; Lardelli, Michael; Khew-Goodall, Yeesim

    2007-09-24

    Epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT. PMID:17893246

  6. 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.

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

    DOE PAGES

    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

  8. 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

  9. Triazine-Based Sequence-Defined Polymers with Side-Chain Diversity and Backbone-Backbone Interaction Motifs.

    PubMed

    Grate, Jay W; Mo, Kai-For; Daily, Michael D

    2016-03-14

    Sequence control in polymers, well-known in nature, encodes structure and functionality. Here we introduce a new architecture, based on the nucleophilic aromatic substitution chemistry of cyanuric chloride, that creates a new class of sequence-defined polymers dubbed TZPs. Proof of concept is demonstrated with two synthesized hexamers, having neutral and ionizable side chains. Molecular dynamics simulations show backbone-backbone interactions, including H-bonding motifs and pi-pi interactions. This architecture is arguably biomimetic while differing from sequence-defined polymers having peptide bonds. The synthetic methodology supports the structural diversity of side chains known in peptides, as well as backbone-backbone hydrogen-bonding motifs, and will thus enable new macromolecules and materials with useful functions. PMID:26865312

  10. HOXB7, a homeodomain protein, is overexpressed in breast cancer and confers epithelial-mesenchymal transition.

    PubMed

    Wu, Xinyan; Chen, Hexin; Parker, Belinda; Rubin, Ethel; Zhu, Tao; Lee, Ji Shin; Argani, Pedram; Sukumar, Saraswati

    2006-10-01

    Epithelial-mesenchymal transition (EMT) is increasingly recognized as a mechanism whereby cells in primary noninvasive tumors acquire properties essential for migration and invasion. Microarray analyses of microdissected epithelial cells from bone metastasis revealed a HOXB7 overexpression that was 3-fold higher than in primary breast carcinomas and 18-fold higher compared with normal breast. This led us to investigate the role of HOXB7 in neoplastic transformation of breast cells. Expression of HOXB7 in both MCF10A and Madin-Darby canine kidney (MDCK) epithelial cells resulted in the acquisition of both phenotypic and molecular attributes typical of EMT. Loss of epithelial proteins, claudin 1 and claudin 7, mislocalization of claudin 4 and E-cadherin, and the expression of mesenchymal proteins, vimentin and alpha-smooth muscle actin, were observed. MDCK cells expressing HOXB7 exhibited properties of migration and invasion. Unlike MDCK vector-transfected cells, MDCK-HOXB7 cells formed highly vascularized tumors in mice. MDCK-HOXB7 cells overexpressed basic fibroblast growth factor (bFGF), had more active forms of both Ras and RhoA proteins, and displayed higher levels of phosphorylation of p44 and p42 mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinases 1 and 2). Effects initiated by HOXB7 were reversed by specific inhibitors of FGF receptor and the Ras-MAPK pathways. These data provide support for a function for HOXB7 in promoting tumor invasion through activation of Ras/Rho pathway by up-regulating bFGF, a known transcriptional target of HOXB7. Reversal of these effects by HOXB7-specific siRNA further suggested that these effects were mediated by HOXB7. Thus, HOXB7 overexpression caused EMT in epithelial cells, accompanied by acquisition of aggressive properties of tumorigenicity, migration, and invasion.

  11. Conformational flexibility of RecA protein filament: transitions between compressed and stretched states.

    PubMed

    Petukhov, Michael; Lebedev, Dmitry; Shalguev, Valery; Islamov, Akhmed; Kuklin, Aleksandr; Lanzov, Vladislav; Isaev-Ivanov, Vladimir

    2006-11-01

    RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations. PMID:16909421

  12. 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

  13. 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

  14. Accumulation of a translation intermediate of D1 protein by light-dark transition in isolated spinach chloroplasts.

    PubMed

    Inagaki, N; Satoh, K

    1992-03-23

    In an in vitro translation experiment using spinach chloroplasts, a novel protein band of about 17.5 kDa appeared by light to dark transition. The protein never accumulated in detectable amounts either in continuous illumination or in continuous darkness. The 17.5 kDa protein accumulated upon light-dark transition, on the other hand, disappeared by the subsequent illumination. Accumulation of the protein in light, however, was observed when stromal level of ATP in chloroplasts was lowered after preillumination by the addition of various chemical compounds which, irrespective of the mode of action, eventually decrease the ATP level, e.g. atrazine, carbonyl-cyanide-m-chlorophenyl hydrazone and glycerate. The dark-accumulated protein was concluded to be a translation intermediate of D1 protein based on the facts that this component precipitates with specific antibodies and is resistant to lysylendopeptidase treatment. The suppression by chloramphenicol of both appearance upon light-dark transition and disappearance by the subsequent illumination of the protein also supported this conclusion. The phenomenon was discussed in terms of pausing in the translation of psbA mRNA.

  15. Deducing the Kinetics of Protein Synthesis In Vivo from the Transition Rates Measured In Vitro

    PubMed Central

    Rudorf, Sophia; Thommen, Michael; Rodnina, Marina V.; Lipowsky, Reinhard

    2014-01-01

    The molecular machinery of life relies on complex multistep processes that involve numerous individual transitions, such as molecular association and dissociation steps, chemical reactions, and mechanical movements. The corresponding transition rates can be typically measured in vitro but not in vivo. Here, we develop a general method to deduce the in-vivo rates from their in-vitro values. The method has two basic components. First, we introduce the kinetic distance, a new concept by which we can quantitatively compare the kinetics of a multistep process in different environments. The kinetic distance depends logarithmically on the transition rates and can be interpreted in terms of the underlying free energy barriers. Second, we minimize the kinetic distance between the in-vitro and the in-vivo process, imposing the constraint that the deduced rates reproduce a known global property such as the overall in-vivo speed. In order to demonstrate the predictive power of our method, we apply it to protein synthesis by ribosomes, a key process of gene expression. We describe the latter process by a codon-specific Markov model with three reaction pathways, corresponding to the initial binding of cognate, near-cognate, and non-cognate tRNA, for which we determine all individual transition rates in vitro. We then predict the in-vivo rates by the constrained minimization procedure and validate these rates by three independent sets of in-vivo data, obtained for codon-dependent translation speeds, codon-specific translation dynamics, and missense error frequencies. In all cases, we find good agreement between theory and experiment without adjusting any fit parameter. The deduced in-vivo rates lead to smaller error frequencies than the known in-vitro rates, primarily by an improved initial selection of tRNA. The method introduced here is relatively simple from a computational point of view and can be applied to any biomolecular process, for which we have detailed information

  16. 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.

  17. 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

  18. The regulated elimination of transit-amplifying cells preserves tissue homeostasis during protein starvation in Drosophila testis

    PubMed Central

    Yang, Heiko; Yamashita, Yukiko M.

    2015-01-01

    How tissues adapt to varying nutrient conditions is of fundamental importance for robust tissue homeostasis throughout the life of an organism, but the underlying mechanisms are poorly understood. Here, we show that Drosophila testis responds to protein starvation by eliminating transit-amplifying spermatogonia (SG) while maintaining a reduced pool of actively proliferating germline stem cells (GSCs). During protein starvation, SG die in a manner that is mediated by the apoptosis of somatic cyst cells (CCs) that encapsulate SG and regulate their development. Strikingly, GSCs cannot be maintained during protein starvation when CC-mediated SG death is inhibited, leading to an irreversible collapse of tissue homeostasis. We propose that the regulated elimination of transit-amplifying cells is essential to preserve stem cell function and tissue homeostasis during protein starvation. PMID:25968311

  19. 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

  20. Conformational transitions in peptides containing two putative alpha-helices of the prion protein.

    PubMed

    Zhang, H; Kaneko, K; Nguyen, J T; Livshits, T L; Baldwin, M A; Cohen, F E; James, T L; Prusiner, S B

    1995-07-21

    Prions are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrPSc). Conversion of the cellular isoform (PrPC) to PrPSc is accompanied by a diminution in the alpha-helical content and an increase in the beta-sheet structure. To investigate the structural basis of this transition, peptide fragments corresponding to Syrian hamster PrP residues 90 to 145 and 109 to 141, which contain the most conserved residues of the prion protein and the first two putative alpha-helical regions in a PrPC model, were studied using infrared spectroscopy and circular dichroism. The peptides could be induced to form alpha-helical structures in aqueous solutions in the presence of organic solvents, such as trifluoroethanol and hexafluoroisopropanol, or detergents, such as sodium dodecyl sulfate and dodecyl phosphocholine. NaCl at physiological concentration or acetonitrile induced the peptides to acquire substantial beta-sheet. The intermolecular nature of the beta-sheet was evident in the formation of rod-shaped polymers as detected by electron microscopy. Resistance to hydrolysis by proteinase K and epitope mapping argue that the beta-sheet structures were formed by the interaction of residues lying between 109 and 141. A similar range of residues was shown by nuclear magnetic resonance spectroscopy to be capable of forming alpha-helices. The alpha-helical structures seem to require a hydrophobic support from either intermolecular interactions or the hydrophobic environment provided by micelles, in agreement with the predicted hydrophobic nature of the packing surface among the four putative helices of PrPC and the outer surfaces of the first two helices. Our results suggest that perturbation of the packing environment of the highly conserved residues is a possible mechanism for triggering the conversion of PrPC to PrPSc where alpha-helices appear to be converted into beta-sheets.

  1. 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.

  2. 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

  3. Nanoscale probing of electron-regulated structural transitions in silk proteins by near-field IR imaging and nano-spectroscopy

    PubMed Central

    Qin, Nan; Zhang, Shaoqing; Jiang, Jianjuan; Corder, Stephanie Gilbert; Qian, Zhigang; Zhou, Zhitao; Lee, Woonsoo; Liu, Keyin; Wang, Xiaohan; Li, Xinxin; Shi, Zhifeng; Mao, Ying; Bechtel, Hans A.; Martin, Michael C.; Xia, Xiaoxia; Marelli, Benedetto; Kaplan, David L.; Omenetto, Fiorenzo G.; Liu, Mengkun; Tao, Tiger H.

    2016-01-01

    Silk protein fibres produced by silkworms and spiders are renowned for their unparalleled mechanical strength and extensibility arising from their high-β-sheet crystal contents as natural materials. Investigation of β-sheet-oriented conformational transitions in silk proteins at the nanoscale remains a challenge using conventional imaging techniques given their limitations in chemical sensitivity or limited spatial resolution. Here, we report on electron-regulated nanoscale polymorphic transitions in silk proteins revealed by near-field infrared imaging and nano-spectroscopy at resolutions approaching the molecular level. The ability to locally probe nanoscale protein structural transitions combined with nanometre-precision electron-beam lithography offers us the capability to finely control the structure of silk proteins in two and three dimensions. Our work paves the way for unlocking essential nanoscopic protein structures and critical conditions for electron-induced conformational transitions, offering new rules to design protein-based nanoarchitectures. PMID:27713412

  4. Nanoscale probing of electron-regulated structural transitions in silk proteins by near-field IR imaging and nano-spectroscopy

    NASA Astrophysics Data System (ADS)

    Qin, Nan; Zhang, Shaoqing; Jiang, Jianjuan; Corder, Stephanie Gilbert; Qian, Zhigang; Zhou, Zhitao; Lee, Woonsoo; Liu, Keyin; Wang, Xiaohan; Li, Xinxin; Shi, Zhifeng; Mao, Ying; Bechtel, Hans A.; Martin, Michael C.; Xia, Xiaoxia; Marelli, Benedetto; Kaplan, David L.; Omenetto, Fiorenzo G.; Liu, Mengkun; Tao, Tiger H.

    2016-10-01

    Silk protein fibres produced by silkworms and spiders are renowned for their unparalleled mechanical strength and extensibility arising from their high-β-sheet crystal contents as natural materials. Investigation of β-sheet-oriented conformational transitions in silk proteins at the nanoscale remains a challenge using conventional imaging techniques given their limitations in chemical sensitivity or limited spatial resolution. Here, we report on electron-regulated nanoscale polymorphic transitions in silk proteins revealed by near-field infrared imaging and nano-spectroscopy at resolutions approaching the molecular level. The ability to locally probe nanoscale protein structural transitions combined with nanometre-precision electron-beam lithography offers us the capability to finely control the structure of silk proteins in two and three dimensions. Our work paves the way for unlocking essential nanoscopic protein structures and critical conditions for electron-induced conformational transitions, offering new rules to design protein-based nanoarchitectures.

  5. 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

  6. 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.

  7. 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

  8. Structural proteins from whelk egg capsule with long range elasticity associated with a solid-state phase transition.

    PubMed

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

    2014-01-13

    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-helical coiled-coil 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) spectroscopy 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.

  9. 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.

  10. 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.

  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. Transition from embryonic to adult epidermis in reptiles occurs by the production of corneous beta-proteins.

    PubMed

    Alibardi, Lorenzo

    2014-01-01

    The adaptation of the epidermis in amniote vertebrates to life on land took place by a drastic change from an embryonic epidermis made of two-four periderm layers to a terrestrial-proof epidermis. This transition occurred by the increase in types and number of specialized corneous proteins coded by genes of the Epidermal Differentiation Complex. The prevalent types of corneous proteins produced in the reptilian epidermis contain a beta-sheet region of high amino acid homology which allows their polymerization into a meshwork of filaments forming the hard corneous material of scales and claws. The present immunogold ultrastructural study shows that this transition occurs with the synthesis of glycine-rich corneous beta-proteins (formerly indicated as beta-keratins) that are added to the initial framework of acidic intermediate filaments produced in the embryonic epidermis of lizards, snake, alligator and turtle. These corneous beta-proteins are accumulated in the transitional and definitive layers of reptilian epidermis formed underneath the transitory two-four layered embryonic epidermis. In the more specialized reptiles capable of shedding the epidermis as a single unit, such as lizards and snakes, special glycine-cysteine rich beta-proteins are initially produced in a single layer immediately formed beneath the embryonic epidermis, the oberhautchen. The latter layer allows the in ovo shedding of the embryonic epidermis in preparation for hatching, and in the following shedding cycles of the adult epidermis. The production of specialized corneous-specific beta-proteins in addition to intermediate filament keratins was probably an essential addition for terrestrial life during the evolution of reptiles into different lineages, including birds. The increase of glycine and cysteine in epidermal proteins enhanced the hydrophobicity, insolubility and mechanical strength of the stratum corneum in these amniotes.

  13. 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

  14. 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.

  15. Doxazosin inhibits retinoblastoma protein phosphorylation and G(1)-->S transition in human coronary smooth muscle cells.

    PubMed

    Kintscher, U; Wakino, S; Kim, S; Jackson, S M; Fleck, E; Hsueh, W A; Law, R E

    2000-05-01

    Previous studies have demonstrated that the alpha(1)-adrenergic receptor antagonist doxazosin (Dox) inhibits multiple mitogenic signaling pathways in human vascular smooth muscle cells. This broad antiproliferative activity of Dox occurs through a novel mechanism unrelated to its blocking the alpha(1)-adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitogen-induced G(1)-->S progression of human coronary artery smooth muscle cells (CASMCs) in a dose-dependent manner, with a maximal reduction of S-phase transition by 88+/-10.5% in 20 ng/mL platelet-derived growth factor and 1 micromol/L insulin (P+I)-stimulated cells (P<0.01 for 10 micromol/L Dox versus P+I alone) and 52+/-18.7% for 10% FBS-induced mitogenesis (P<0.05 for 10 micromol/L Dox versus 10% FBS alone). Inhibition of G(1) exit by Dox was accompanied by a significant blockade of retinoblastoma protein (Rb) phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G(1) arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CASMCs to progress through G(1) and enter the S phase. E2F-mediated G(1) exit was not affected by Dox, suggesting that it targets events upstream from Rb hyperphosphorylation. Downregulation of the cyclin-dependent kinase inhibitory protein p27 is important for maximal activation of G(1) cyclin/cyclin-dependent kinase holoenzymes to overcome the cell cycle inhibitory activity of Rb. In Western blot analysis, p27 levels decreased after mitogenic stimulation (after P+I, 43+/-1.8% of quiescent cells [P<0.01 versus quiescent cells]; after 10% FBS, 55+/-7.7% of quiescent cells [P<0. 05 versus quiescent cells]), whereas the addition of Dox (10 micromol/L) markedly attenuated its downregulation (after P+I, 90+/-8.3% of quiescent cells [P<0.05 versus P+I alone]; after 10% FBS, 78+/-8.3% of quiescent cells [P<0.05 versus 10% FBS alone]). Furthermore, Dox inhibited cyclin A expression, an E2F regulated gene that is essential for cell cycle

  16. Photoacids as a new fluorescence tool for tracking structural transitions of proteins: following the concentration-induced transition of bovine serum albumin.

    PubMed

    Amdursky, Nadav

    2015-12-21

    Spectroscopy-based techniques for assessing structural transitions of proteins follow either an intramolecular chromophore, as in absorption-based circular dichroism (CD) or fluorescence-based tryptophan emission, or an intermolecular chromophore such as fluorescent probes. Here a new fluorescent probe method to probe the structural transition of proteins by photoacids is presented, which has a fundamentally different photo-physical origin to that of common fluorescent probes. Photoacids are molecules that release a proton upon photo-excitation. By following the steady-state and time-resolved emission of the protonated and de-protonated species of the photoacid we probe the environment of its binding site in bovine serum albumin (BSA) in a wide range of weight concentrations (0.001-8%). We found a unique concentration-induced structural transition of BSA at pH2 and at concentrations of >0.75%, which involves the exposure of its hydrophobic core to the solution. We confirm our results with the common tryptophan emission method, and show that the use of photoacids can result in a much more sensitive tool. We also show that common fluorescent probes and the CD methodologies have fundamental restrictions that limit their use in a concentration-dependent study. The use of photoacids is facile and requires only a fluorospectrometer (and preferably, but not mandatorily, a time-resolution emission system). The photoacid can be either non-covalently (as in this study) or covalently attached to the molecule, and can be readily employed to follow the local environment of numerous (bio-)systems. PMID:26573990

  17. 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.

  18. Dynamics of protein folding: probing the kinetic network of folding-unfolding transitions with experiment and theory.

    PubMed

    Buchner, Ginka S; Murphy, Ronan D; Buchete, Nicolae-Viorel; Kubelka, Jan

    2011-08-01

    The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less "downhill" folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding-unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at

  19. Improvement of the treatment of loop structures in the UNRES force field by inclusion of coupling between backbone- and side-chain-local conformational states

    PubMed Central

    Baranowski, Maciej; Ołldziej, Stanisław; Scheraga, Harold A.; Liwo, Adam; Czaplewski, Cezary

    2013-01-01

    The UNited RESidue (UNRES) coarse-grained model of polypeptide chains, developed in our laboratory, enables us to carry out millisecond-scale molecular-dynamics simulations of large proteins effectively. It performs well in ab initio predictions of protein structure, as demonstrated in the last Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP10). However, the resolution of the simulated structure is too coarse, especially in loop regions, which results from insufficient specificity of the model of local interactions. To improve the representation of local interactions, in this work we introduced new side-chain-backbone correlation potentials, derived from a statistical analysis of loop regions of 4585 proteins. To obtain sufficient statistics, we reduced the set of amino-acid-residue types to five groups, derived in our earlier work on structurally optimized reduced alphabets, based on a statistical analysis of the properties of amino-acid structures. The new correlation potentials are expressed as one-dimensional Fourier series in the virtual-bond-dihedral angles involving side-chain centroids. The weight of these new terms was determined by a trial-and-error method, in which Multiplexed Replica Exchange Molecular Dynamics (MREMD) simulations were run on selected test proteins. The best average root-mean-square deviations (RMSDs) of the calculated structures from the experimental structures below the folding-transition temperatures were obtained with the weight of the new side-chain-backbone correlation potentials equal to 0.57. The resulting conformational ensembles were analyzed in detail by using the Weighted Histogram Analysis Method (WHAM) and Ward's minimum-variance clustering. This analysis showed that the RMSDs from the experimental structures dropped by 0.5 Å on average, compared to simulations without the new terms, and the deviation of individual residues in the loop region of the computed

  20. Structural mimicry of the α-helix in aqueous solution with an isoatomic α/β/γ-peptide backbone.

    PubMed

    Sawada, Tomohisa; Gellman, Samuel H

    2011-05-18

    Artificial mimicry of α-helices offers a basis for development of protein-protein interaction antagonists. Here we report a new type of unnatural peptidic backbone, containing α-, β-, and γ-amino acid residues in an αγααβα repeat pattern, for this purpose. This unnatural hexad has the same number of backbone atoms as a heptad of α residues. Two-dimensional NMR data clearly establish the formation of an α-helix-like conformation in aqueous solution. The helix formed by our 12-mer α/β/γ-peptide is considerably more stable than the α-helix formed by an analogous 14-mer α-peptide, presumably because of the preorganized β and γ residues employed.

  1. Allosteric Transitions Direct Protein Tagging by PafA, the Prokaryotic Ubiquitin-like Protein (Pup) Ligase*

    PubMed Central

    Ofer, Naomi; Forer, Nadav; Korman, Maayan; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2013-01-01

    Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery. PMID:23471967

  2. Allosteric transitions direct protein tagging by PafA, the prokaryotic ubiquitin-like protein (Pup) ligase.

    PubMed

    Ofer, Naomi; Forer, Nadav; Korman, Maayan; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2013-04-19

    Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery. PMID:23471967

  3. SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament.

    PubMed

    Hu, Huiqing; Zhou, Qing; Li, Ziyin

    2015-12-18

    The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.

  4. SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament.

    PubMed

    Hu, Huiqing; Zhou, Qing; Li, Ziyin

    2015-12-18

    The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei. PMID:26504079

  5. Extracting the multiscale backbone of complex weighted networks

    PubMed Central

    Serrano, M. Ángeles; Boguñá, Marián; Vespignani, Alessandro

    2009-01-01

    A large number of complex systems find a natural abstraction in the form of weighted networks whose nodes represent the elements of the system and the weighted edges identify the presence of an interaction and its relative strength. In recent years, the study of an increasing number of large-scale networks has highlighted the statistical heterogeneity of their interaction pattern, with degree and weight distributions that vary over many orders of magnitude. These features, along with the large number of elements and links, make the extraction of the truly relevant connections forming the network's backbone a very challenging problem. More specifically, coarse-graining approaches and filtering techniques come into conflict with the multiscale nature of large-scale systems. Here, we define a filtering method that offers a practical procedure to extract the relevant connection backbone in complex multiscale networks, preserving the edges that represent statistically significant deviations with respect to a null model for the local assignment of weights to edges. An important aspect of the method is that it does not belittle small-scale interactions and operates at all scales defined by the weight distribution. We apply our method to real-world network instances and compare the obtained results with alternative backbone extraction techniques. PMID:19357301

  6. 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

  7. 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.

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

    PubMed

    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. PMID:23656161

  9. 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.

  10. Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

    DOE PAGES

    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

  11. High-throughput backbone resonance assignment of small 13C, 15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J ( 1H, 13C): HNCACB codedHAHB

    NASA Astrophysics Data System (ADS)

    Pegan, Scott; Kwiatkowski, Witek; Choe, Senyon; Riek, Roland

    2003-12-01

    The proposed three-dimensional triple-resonance experiment HNCACB codedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13C α, 13C β, 1H α, and 1H β. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1H α and 1H β chemical shifts are then coded in the line shape of the cross-peaks of 13C α, 13C β along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACB codedHAHB experiment was applied to ˜85% labeled 13C, 15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.

  12. 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

  13. Lost in Transit: Long-Distance Trafficking and Phloem Unloading of Protein Signals in Arabidopsis Homografts[OPEN

    PubMed Central

    Gustin, Marie-Paule; Molnar, Attila; Oparka, Karl J.

    2016-01-01

    In addition to moving sugars and nutrients, the phloem transports many macromolecules. While grafting and aphid stylectomy experiments have identified many macromolecules that move in the phloem, the functional significance of phloem transport of these remains unclear. To gain insight into protein trafficking, we micrografted Arabidopsis thaliana scions expressing GFP-tagged chloroplast transit peptides under the 35S promoter onto nontransgenic rootstocks. We found that plastids in the root tip became fluorescent 10 d after grafting. We obtained identical results with the companion cell-specific promoter SUC2 and with signals that target proteins to peroxisomes, actin, and the nucleus. We were unable to detect the respective mRNAs in the rootstock, indicating extensive movement of proteins in the phloem. Outward movement from the root protophloem was restricted to the pericycle-endodermis boundary, identifying plasmodesmata at this interface as control points in the exchange of macromolecules between stele and cortex. Intriguingly, signals directing proteins to the endoplasmic reticulum and Golgi apparatus from membrane-bound ribosomes were not translocated to the root. It appears that many organelle-targeting sequences are insufficient to prevent the loss of their proteins into the translocation stream. Thus, nonspecific loss of proteins from companion cells to sieve elements may explain the plethora of macromolecules identified in phloem sap. PMID:27600534

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

    DOE PAGES

    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

  15. 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.

  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. 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.

  18. Supramolecular organization of the repetitive backbone unit of the Streptococcus pneumoniae pilus.

    PubMed

    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

    2010-06-15

    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 A 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.

  19. 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

  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. 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.

  2. 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.

  3. Plastid localization of the PEND protein is mediated by a noncanonical transit peptide.

    PubMed

    Terasawa, Kimihiro; Sato, Naoki

    2009-03-01

    Plastid envelope DNA-binding protein (PEND) is a DNA-binding protein with a chloroplast basic region-zipper domain at its N-terminus and a transmembrane domain at its C-terminus. The localization of PEND to the inner envelope membrane was demonstrated in a targeting experiment using isolated membranes and green fluorescent protein-tagged fusion proteins. An N-terminal sequence analysis showed that the presequence is 15 amino acids long; however, based on neural network-based prediction tools, this short peptide is not predicted to be a chloroplast-targeting sequence. In the present study we confirmed, by the digestion of intact chloroplasts, that PEND is located in the envelope membrane. We then demonstrated that the N-terminal 88-amino acid sequence is sufficient for plastid import in vitro. The transient expression of green fluorescent protein-tagged fusion proteins revealed that neither the N-terminal 29-amino acid sequence nor the 16-amino acid sequence directed green fluorescent protein to chloroplasts, but that the N-terminal 66-amino acid sequence was sufficient for correct targeting. These results suggest that targeting of PEND to the chloroplast requires both the presequence and the basic region, whereas postimport processing cleaves only the presequence. Interestingly, deletion of the presequence in the green fluorescent protein-tagged 88-amino acid construct resulted in targeting to the nucleus. This raises the possibility of plastid-to-nuclear signal transduction by the relocalization of PEND. PMID:19220850

  4. 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.

  5. 8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme

    PubMed Central

    Kiryutin, Alexey S.; Kasymov, Rustem D.; Petrova, Darya V.; Endutkin, Anton V.; Popov, Alexander V.; Yurkovskaya, Alexandra V.; Fedechkin, Stanislav O.; Brockerman, Jacob A.; Zharkov, Dmitry O.; Smirnov, Serge L.

    2016-01-01

    8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew—Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar—phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein—DNA interface. PMID:27749894

  6. Resistance of Feynman diagrams and the percolation backbone dimension.

    PubMed

    Janssen, H K; Stenull, O; Oerding, K

    1999-06-01

    We present an alternative view of Feynman diagrams for the field theory of random resistor networks, in which the diagrams are interpreted as being resistor networks themselves. This simplifies the field theory considerably as we demonstrate by calculating the fractal dimension D(B) of the percolation backbone to three loop order. Using renormalization group methods we obtain D(B)=2+epsilon/21-172epsilon(2)/9261+2epsilon(3)[-74 639+22 680zeta(3)]/4 084 101, where epsilon=6-d with d being the spatial dimension and zeta(3)=1.202 057... .

  7. Protein-transitions in and out of the dough matrix in wheat flour mixing.

    PubMed

    Wang, Xiaolong; Appels, Rudi; Zhang, Xiaoke; Bekes, Ferenc; Torok, Kitti; Tomoskozi, Sandor; Diepeveen, Dean; Ma, Wujun; Islam, Shahidul

    2017-02-15

    Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour. PMID:27664670

  8. Protein-transitions in and out of the dough matrix in wheat flour mixing.

    PubMed

    Wang, Xiaolong; Appels, Rudi; Zhang, Xiaoke; Bekes, Ferenc; Torok, Kitti; Tomoskozi, Sandor; Diepeveen, Dean; Ma, Wujun; Islam, Shahidul

    2017-02-15

    Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour.

  9. 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

  10. Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent.

    PubMed

    Renner, C; Cramer, J; Behrendt, R; Moroder, L

    2000-12-01

    It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to

  11. SOP‐GPU: influence of solvent‐induced hydrodynamic interactions on dynamic structural transitions in protein assemblies

    PubMed Central

    Alekseenko, Andrey; Kononova, Olga; Kholodov, Yaroslav; Marx, Kenneth A.

    2016-01-01

    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 (102–105 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 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. PMID:27015749

  12. 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.

  13. Carbenoxolone induces permeability transition pore opening in rat mitochondria via the translocator protein TSPO and connexin43.

    PubMed

    Azarashvili, Tamara; Baburina, Yulia; Grachev, Dmitry; Krestinina, Olga; Papadopoulos, Vassilios; Lemasters, John J; Odinokova, Irina; Reiser, Georg

    2014-09-15

    Ca(2+)-induced permeability transition pore (mPTP) opening in isolated rat brain mitochondria is promoted through targeting of connexin43. After a threshold Ca(2+) load, mitochondrial membrane potential drops and efflux of accumulated Ca(2+) from the mitochondrial matrix occurs, indicating the mPTP opening. Specific antibodies were used to assess the role of the translocator protein (18kDa; TSPO) and connexin43 in swelling of isolated rat liver and brain mitochondria induced by carbenoxolone and the endogenous TSPO ligand protoporphyrin IX. Mitochondrial membrane potential, Ca(2+) transport and oxygen consumption were determined using selective electrodes. All the parameters were detected simultaneously in a chamber with the selective electrodes. The phosphorylation state of mitochondrial protein targets was assessed. We report that Ca(2+)-induced mitochondrial swelling was strengthened in the presence of both carbenoxolone and protoporphyrin IX. The carbenoxolone- and protoporphyrin IX-accelerated mPTP induction in brain mitochondria was completely prevented by antibodies specific for the mitochondrial translocator protein (TSPO). The anti-TSPO antibodies were more effective than anti-сonnexin43 antibodies. Moreover, carbenoxolone-stimulated phosphorylation of mitochondrial proteins was inhibited by anti-TSPO antibodies. Taken together, the data suggests that, in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO.

  14. 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

  15. Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR.

    PubMed Central

    Yi, Q.; Baker, D.

    1996-01-01

    We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates. PMID:8762137

  16. 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.

  17. 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.

  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. SHP2 positively regulates TGFβ1-induced epithelial-mesenchymal transition modulated by its novel interacting protein Hook1.

    PubMed

    Li, Shuomin; Wang, Linrun; Zhao, Qingwei; Liu, Yu; He, Lingjuan; Xu, Qinqin; Sun, Xu; Teng, Li; Cheng, Hongqiang; Ke, Yuehai

    2014-12-01

    The epithelial-mesenchymal transition (EMT) is an essential process for embryogenesis. It also plays a critical role in the initiation of tumor metastasis. Src homology 2 (SH2)-domain containing protein-tyrosine phosphatase-2 (SHP2) is a ubiquitously expressed protein-tyrosine phosphatase and is mutated in many tumors. However, its functional role in tumor metastasis remains largely unknown. We found that TGFβ1-induced EMT in lung epithelial A549 cells was partially blocked when SHP2 was decreased by transfected siRNA. The constitutively active form (E76V) promoted EMT while the phosphatase-dead mutation (C459S) and the SHP2 inhibitor PHPS1 blocked EMT, which further demonstrated that the phosphatase activity of SHP2 was required for promoting TGFβ1-induced EMT. Using the protein-tyrosine phosphatase domain of SHP2 as bait, we identified a novel SHP2-interacting protein Hook1. Hook1 was down-regulated during EMT in A549 cells. Overexpression of Hook1 inhibited EMT while knockdown of Hook1 promoted EMT. Moreover, both the protein-tyrosine phosphatase domain and N-terminal SH2 domain of SHP2 directly interacted with Hook1. Down-regulation of Hook1 increased SHP2 activity. These results suggested that Hook1 was an endogenous negative regulator of SHP2 phosphatase activity. Our data showed that the protein-tyrosine phosphatase SHP2 was involved in the process of EMT and Hook1 repressed EMT by regulating the activation of SHP2. SHP2-Hook1 complex may play important roles in tumor metastases by regulating EMT in cancer cells.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. ATF6 pathway of unfolded protein response mediates advanced oxidation protein product-induced hypertrophy and epithelial-to-mesenchymal transition in HK-2 cells.

    PubMed

    Tang, Xun; Liang, Xiujie; Li, Minhui; Guo, Tingting; Duan, Na; Wang, Yue; Rong, Guang; Yang, Lei; Zhang, Shaojie; Zhang, Jun

    2015-09-01

    Advanced oxidation protein products (AOPPs) accelerate the progression of chronic kidney disease. We previously demonstrated that AOPPs induce hypertrophy and epithelial-to-mesenchymal transition (EMT) in human proximal tubular cells (HK-2 cells) through induction of endoplasmic reticulum (ER) stress. However, which pathway of unfolded protein response (UPR) induced by ER stress plays crucial roles in this process remains unclear. In this study, we investigated the roles of the protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) pathways of UPR in this process in HK-2 cells. AOPP treatment induced the overexpression of cleaved ATF6 and spliced form of X-box binding protein-1, and induced the phosphorylation of PERK, eukaryotic translation initiation factor 2α and IRE1. Furthermore, silencing of ATF6 increased E-cadherin and zonula occludens-1 expression, lowered the expression of vimentin, and downregulated total protein content, whereas knockdown of PERK or IRE1 resulted in no difference compared with the scramble siRNA-transfected cells. AOPP-induced phosphorylation of Src, which was reproduced by thapsigargin, an inducer of ER stress, was partly reversed by salubrinal, an inhibitor of ER stress. Furthermore, the Src inhibitor saracatinib effectively blocked AOPP-induced phosphorylation of Src, activation of ER stress, hypertrophy, and EMT in HK-2 cells. Collectively, our results indicate that AOPPs induce the PERK, ATF6, and IRE1 pathways of UPR, and the ATF6 pathway rather than the other two pathways mediates AOPP-induced HK-2-cell hypertrophy and EMT. We also suggest that the ER stress involved in this process is likely mediated by the activation of Src kinase. PMID:26045172

  5. Marburg virus VP35 can both fully coat the backbone and cap the ends of dsRNA for interferon antagonism.

    PubMed

    Bale, Shridhar; Julien, Jean-Philippe; Bornholdt, Zachary A; Kimberlin, Christopher R; Halfmann, Peter; Zandonatti, Michelle A; Kunert, John; Kroon, Gerard J A; Kawaoka, Yoshihiro; MacRae, Ian J; Wilson, Ian A; Saphire, Erica Ollmann

    2012-09-01

    Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.

  6. 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

  7. Dual-functional ROMP-based betaines: effect of hydrophilicity and backbone structure on nonfouling properties.

    PubMed

    Colak, Semra; Tew, Gregory N

    2012-01-10

    Foundational materials for nonfouling coatings were designed and synthesized from a series of novel dual-functional zwitterionic polymers, Poly[NRZI], which were easily obtained via ring-opening metathesis polymerization (ROMP) followed by a single step transformation of the cationic precursor, Poly[NR(+)], to the zwitterion, Poly[NRZI]. The resulting unique dual-functional structure contained the anion and the cation within the same repeat unit but on separate side chains, enabling the hydrophilicity of the system to be tuned at the repeat unit level. These dual-functional zwitterionic polymers were specifically designed to investigate the impact of structural changes, including the backbone, hydrophilicity, and charge, on the overall nonfouling properties. To evaluate the importance of backbone structure, and as a direct comparison to previously studied methacrylate-based betaines, norbornene-based carbo- and sulfobetaines (Poly[NCarboZI] and Poly[NSulfoZI]) as well as a methacrylate-based sulfobetaine (Poly[MASulfoZI]) were synthesized. These structures contain the anion-cation pairs on the same side chain. Nonfouling coatings were prepared from copolymers, composed of the zwitterionic/cationic precursor monomer and an ethoxysilane-containing monomer. The coatings were evaluated by using protein adsorption studies, which clearly indicated that the overall hydrophilicity has a major influence on the nonfouling character of the materials. The most hydrophilic coating, from the oligoethylene glycol (OEG)-containing dual-functional betaine, Poly[NOEGZI-co-NSi], showed the best resistance to nonspecific protein adsorption (Γ(FIB) = 0.039 ng/mm(2)). Both norbornene-based polymers systems, Poly[NSulfoZI] and Poly[NCarboZI], were more hydrophilic and thus more resistant to protein adsorption than the methacrylate-based Poly[MASulfoZI]. Comparing the protein resistance of the dual-functional zwitterionic coatings, Poly[NRZI-co-NSi], to that of their cationic

  8. GASA: a graph-based automated NMR backbone resonance sequential assignment program.

    PubMed

    Wan, Xiang; Lin, Guohui

    2007-04-01

    The success in backbone resonance sequential assignment is fundamental to three dimensional protein structure determination via Nuclear Magnetic Resonance (NMR) spectroscopy. Such a sequential assignment can roughly be partitioned into three separate steps: grouping resonance peaks in multiple spectra into spin systems, chaining the resultant spin systems into strings, and assigning these strings to non-overlapping consecutive amino acid residues in the target protein. Separately dealing with these three steps has been adopted in many existing assignment programs, and it works well on protein NMR data with close-to-ideal quality, while only moderately or even poorly on most real protein datasets, where noises as well as data degeneracies occur frequently. We propose in this work to partition the sequential assignment not by physical steps, but only virtual steps, and use their outputs to cross validate each other. The novelty lies in the places, where the ambiguities at the grouping step will be resolved in finding the highly confident strings at the chaining step, and the ambiguities at the chaining step will be resolved by examining the mappings of strings at the assignment step. In this way, all ambiguities at the sequential assignment will be resolved globally and optimally. The resultant assignment program is called Graph-based Approach for Sequential Assignment (GASA), which has been compared to several recent similar developments including PACES, RANDOM, MARS, and RIBRA. The performance comparisons with these works demonstrated that GASA is more promising for practical use.

  9. 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

  10. The Thermal Structural Transition of Alpha-Crystallin Modulates Subunit Interactions and Increases Protein Solubility

    PubMed Central

    Maulucci, Giuseppe; De Spirito, Marco; Arcovito, Giuseppe; Papi, Massimiliano

    2012-01-01

    Background Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract), is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K). Methods/Results To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ) and the interfacial tension (γ) of the aggregating phase, that characterize subunit interactions. Conclusions/General Significance The decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing. PMID:22347398

  11. 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

  12. 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

  13. Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone.

    PubMed

    Gui, Meng; Song, Juyi; Zhang, Lu; Wang, Shun; Wu, Ruiyun; Ma, Changwei; Li, Pinglan

    2015-06-01

    Chondroitin sulfates (CSs) were extracted from sturgeon skull and backbone, and their chemical composition, anticoagulant, anti-platelet and thrombolysis activities were evaluated. The average molecular weights of CS from sturgeon skull and backbone were 38.5kDa and 49.2kDa, respectively. Disaccharide analysis indicated that the sturgeon backbone CS was primarily composed of disaccharide monosulfated in position four of the GalNAc (37.8%) and disaccharide monosulfated in position six of the GalNAc (59.6%) while sturgeon skull CS was primarily composed of nonsulfated disaccharide (74.2%). Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS. Sturgeon backbone CS could significantly prolong activated partial thromboplastin time (APTT) and thrombin time (TT), inhibited ADP-induced platelet aggregation and dissolved platelet plasma clots in vitro. The results suggested that sturgeon backbone CS can be explored as a functional food with antithrombotic function.

  14. Backbones of evolutionary history test biodiversity theory for microbes.

    PubMed

    O'Dwyer, James P; Kembel, Steven W; Sharpton, Thomas J

    2015-07-01

    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.

  15. 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.

  16. 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

  17. 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

  18. [Dynamics of electron-conformational transitions in proteins and physical mechanisms of biomacromolecule function].

    PubMed

    Shaĭtan, K V

    1992-01-01

    The proteins can be considered as a microheterogeneous structured media possessing memory and feedback properties. The conformational energy surface depends on the chemical states of protein groups. Conformational motions are local diffusion with relaxation times much longer than vibrational relaxation times in condensed media. Owing to the hierarchy of relaxation times chemical reaction rates depend on conformation parametrically. Regulation of functional activity by conformational mobility is accomplished via transmission of information in the form of changes in the distribution functions of separate groups along the conformational substates. The interpretation of drastic effects on conformational mobility needs super-stochastic approaches. A possible mechanism of sharp conformational change are discussed in terms of the catastrophe theory.

  19. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

    PubMed

    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

    2015-03-01

    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

  20. Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma.

    PubMed

    Singh, P K; Srivastava, Anupam K; Rath, S K; Dalela, D; Goel, M M; Bhatt, M L B

    2015-01-01

    Bladder cancer (BC) is one among the most common and lethal urothelial malignancies worldwide. The expression of cancer-testis (CT) antigens in some tumours and restricted expression among normal tissues make CT antigens as attractive vaccine targets. In this context, we evaluated Centrosomal protein 55 kDa (CEP55), which is specifically expressed in normal human testis and various malignancies. Until the expression pattern of CEP55 in transitional cell carcinoma (TCC) of human urinary bladder and its clinical significance are not known. The aim of the present study is to evaluate mRNA/protein expression of CEP55 in TCCs of urinary bladder and correlate its expression with the clinicopathological characteristics of BC patients. In this study, the methods of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to investigate mRNA/protein expression of CEP55 in TCC. Independent Student's t test, ANOVA and Chi-square (χ(2)) were used to analyze the data statistically. We observed CEP55 mRNA overexpression in testis and 48.7% of BC patients. Relative mean fold expression of CEP55 mRNA was found to be significantly (p<0.01) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (7.88±3.88 vs. 4.75±2.30, p=0.01). CEP55 protein expression was evaluated using IHC and cytoplasmic staining pattern was recorded in formalin fixed, paraffin-embedded (FFPE) bladder tumour tissues. No significant difference was observed in protein expression of CEP55 between the two groups (NMIBC and MIBC patients) (72.2% vs. 69.0%, p=0.774). No significant protein expression of CEP55 was observed among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Our study results suggest that CEP55 mRNA/protein expression was observed is specific to TCC of human urinary bladder and might be used as a diagnostic biomarker and vaccine target in development

  1. 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.

  2. Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.

    PubMed

    D'Amours, Olivier; Frenette, Gilles; Bordeleau, Louis-Jean; Allard, Nancy; Leclerc, Pierre; Blondin, Patrick; Sullivan, Robert

    2012-10-01

    Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

  3. Comparison of the backbone dynamics of a natural and a consensus designed 3-TPR domain.

    PubMed

    Jarymowycz, Virginia A; Cortajarena, Aitziber L; Regan, Lynne; Stone, Martin J

    2008-07-01

    The tetratricopeptide repeat (TPR) is a 34-amino acid helix-turn-helix motif that occurs in tandem arrays in numerous proteins. Here we compare the backbone dynamics of a natural 3-repeat TPR domain, from the protein UBP, with the behavior of a designed protein CTPR3, which consists of three identical consensus TPR units. Although the three tandem TPR repeats in both CTPR3 and UBP behave as a single unit, with no evidence of independent repeat motions, the data indicate that certain positions in UBP are significantly more flexible than are the corresponding positions in CTPR3. Most of the dynamical changes occur at or adjacent to positions that are involved in intra-repeat packing interactions. These observations lead us to suggest that the three-TPR domain of UBP does not incorporate optimized packing, compared to that seen in the idealized CTPR. The natural TPR domain is not only less stable overall than CTPR3, but also presents increased local flexibility at the positions where the sequences differs from the conserved consensus.

  4. An avian live attenuated master backbone for potential use in epidemic and pandemic influenza vaccines.

    PubMed

    Hickman, Danielle; Hossain, Md Jaber; Song, Haichen; Araya, Yonas; Solórzano, Alicia; Perez, Daniel R

    2008-11-01

    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.

  5. Peptide backbone cleavage by α-amidation is enhanced at methionine residues.

    PubMed

    Hellwig, Michael; Löbmann, Katja; Orywol, Tom

    2015-01-01

    Cleavage reactions at backbone loci are one of the consequences of oxidation of proteins and peptides. During α-amidation, the Cα -N bond in the backbone is cleaved under formation of an N-terminal peptide amide and a C-terminal keto acyl peptide. On the basis of earlier works, a facilitation of α-amidation by the thioether group of adjacent methionine side chains was proposed. This reaction was characterized by using benzoyl methionine and benzoyl alanyl methionine as peptide models. The decomposition of benzoylated amino acids (benzoyl-methionine, benzoyl-alanine, and benzoyl-methionine sulfoxide) to benzamide in the presence of different carbohydrate compounds (reducing sugars, Amadori products, and reductones) was studied during incubation for up to 48 h at 80 °C in acetate-buffered solution (pH 6.0). Small amounts of benzamide (0.3-1.5 mol%) were formed in the presence of all sugars and from all benzoylated species. However, benzamide formation was strongly enhanced, when benzoyl methionine was incubated in the presence of reductones and Amadori compounds (3.5-4.2 mol%). The reaction was found to be intramolecular, because α-amidation of a similar 4-methylbenzoylated amino acid was not enhanced in the presence of benzoyl-methionine and carbohydrate compounds. In the peptide benzoyl-alanyl-methionine, α-amidation at the methionine residue is preferred over α-amidation at the benzoyl peptide bond. We propose here a mechanism for the enhancement of α-amidation at methionine residues.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Depth dependent dynamics in the hydration shell of a protein

    NASA Astrophysics Data System (ADS)

    Servantie, J.; Atilgan, C.; Atilgan, A. R.

    2010-08-01

    We study the dynamics of hydration water/protein association in folded proteins using lysozyme and myoglobin as examples. Extensive molecular dynamics simulations are performed to identify underlying mechanisms of the dynamical transition that corresponds to the onset of amplified atomic fluctuations in proteins. The results indicate that the number of water molecules within a cutoff distance of each residue scales linearly with protein depth index and is not affected by the local dynamics of the backbone. Keeping track of the water molecules within the cutoff sphere, we observe an effective residence time, scaling inversely with depth index at physiological temperatures while the diffusive escape is highly reduced below the transition. A depth independent orientational memory loss is obtained for the average dipole vector of the water molecules within the sphere when the protein is functional. While below the transition temperature, the solvent is in a glassy state, acting as a solid crust around the protein, inhibiting any large scale conformational fluctuations. At the transition, most of the hydration shell unfreezes and water molecules collectively make the protein more flexible.

  15. 1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

    PubMed Central

    Feher, V. A.; Zapf, J. W.; Hoch, J. A.; Dahlquist, F. W.; Whiteley, J. M.; Cavanagh, J.

    1995-01-01

    Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54. PMID:8528078

  16. Microenvironments created by liquid-liquid phase transition control the dynamic distribution of bacterial division FtsZ protein

    PubMed Central

    Monterroso, Begoña; Zorrilla, Silvia; Sobrinos-Sanguino, Marta; Keating, Christine D.; Rivas, Germán

    2016-01-01

    The influence of membrane-free microcompartments resulting from crowding-induced liquid/liquid phase separation (LLPS) on the dynamic spatial organization of FtsZ, the main component of the bacterial division machinery, has been studied using several LLPS systems. The GTP-dependent assembly cycle of FtsZ is thought to be crucial for the formation of the septal ring, which is highly regulated in time and space. We found that FtsZ accumulates in one of the phases and/or at the interface, depending on the system composition and on the oligomerization state of the protein. These results were observed both in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets. The visualization of the droplets revealed that both the location and structural arrangement of FtsZ filaments is determined by the nature of the LLPS. Relocation upon depolymerization of the dynamic filaments suggests the protein may shift among microenvironments in response to changes in its association state. The existence of these dynamic compartments driven by phase transitions can alter the local composition and reactivity of FtsZ during its life cycle acting as a nonspecific modulating factor of cell function. PMID:27725777

  17. Thermodynamics and kinetics of protein folding on the ribosome: Alteration in energy landscapes, denatured state, and transition state ensembles

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward; Vendruscolo, Michele; Dobson, Christopher

    2010-03-01

    In vitro experiments examining cotranslational folding utilize ribosome-nascent chain complexes (RNCs) in which the nascent chain is stalled at different points of its biosynthesis on the ribosome. We investigate the thermodynamics, kinetics, and structural properties of RNCs containing five different globular and repeat proteins stalled at ten different nascent chain lengths using coarse grained replica exchange simulations. We find that when the proteins are stalled near the ribosome exit tunnel opening they exhibit altered folding coopserativity, quantified by the van't Hoff enthalpy criterion; a significantly altered denatured state ensemble, in terms of Rg and shape parameters (Rg tensor); and the appearance of partially folded intermediates during cotranslation, evidenced by the appearance of a third basin in the free energy profile. These trends are due in part to excluded volume (crowding) interactions between the ribosome and nascent chain. We perform in silico temperature-jump experiments on the RNCs and examine nascent chain folding kinetics and structural changes in the transition state ensemble at various stall lengths.

  18. 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

  19. 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

  20. The microtubule destabilizing protein stathmin controls the transition from dividing neuronal precursors to postmitotic neurons during adult hippocampal neurogenesis.

    PubMed

    Boekhoorn, Karin; van Dis, Vera; Goedknegt, Erika; Sobel, André; Lucassen, Paul J; Hoogenraad, Casper C

    2014-12-01

    The hippocampus is one of the two areas in the mammalian brain where adult neurogenesis occurs. Adult neurogenesis is well known to be involved in hippocampal physiological functions as well as pathophysiological conditions. Microtubules (MTs), providing intracellular transport, stability, and transmitting force, are indispensable for neurogenesis by facilitating cell division, migration, growth, and differentiation. Although there are several examples of MT-stabilizing proteins regulating different aspects of adult neurogenesis, relatively little is known about the function of MT-destabilizing proteins. Stathmin is such a MT-destabilizing protein largely restricted to the CNS, and in contrast to its developmental family members, stathmin is also expressed at significant levels in the adult brain, notably in areas involved in adult neurogenesis. Here, we show an important role for stathmin during adult neurogenesis in the subgranular zone of the mouse hippocampus. After carefully mapping stathmin expression in the adult dentate gyrus (DG), we investigated its role in hippocampal neurogenesis making use of stathmin knockout mice. Although hippocampus development appears normal in these animals, different aspects of adult neurogenesis are affected. First, the number of proliferating Ki-67+ cells is decreased in stathmin knockout mice, as well as the expression of the immature markers Nestin and PSA-NCAM. However, newborn cells that do survive express more frequently the adult marker NeuN and have a more mature morphology. Furthermore, our data suggest that migration in the DG might be affected. We propose a model in which stathmin controls the transition from neuronal precursors to early postmitotic neurons.

  1. Fission yeast nucleolar protein Dnt1 regulates G2/M transition and cytokinesis by downregulating Wee1 kinase.

    PubMed

    Yu, Zhi-Yong; Zhang, Meng-Ting; Wang, Gao-Yuan; Xu, Dan; Keifenheim, Daniel; Franco, Alejandro; Cansado, Jose; Masuda, Hirohisa; Rhind, Nick; Wang, Yamei; Jin, Quan-Wen

    2013-11-01

    Cytokinesis involves temporally and spatially coordinated action of the cell cycle, cytoskeletal and membrane systems to achieve separation of daughter cells. The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Previously, we have shown that in fission yeast, the nucleolar protein Dnt1 negatively regulates the SIN pathway in a manner that is independent of the Cdc14-family phosphatase Clp1/Flp1, but how Dnt1 modulates this pathway has remained elusive. By contrast, it is clear that its budding yeast relative, Net1/Cfi1, regulates the homologous MEN signaling pathway by sequestering Cdc14 phosphatase in the nucleolus before mitotic exit. In this study, we show that dnt1(+) positively regulates G2/M transition during the cell cycle. By conducting epistasis analyses to measure cell length at septation in double mutant (for dnt1 and genes involved in G2/M control) cells, we found a link between dnt1(+) and wee1(+). Furthermore, we showed that elevated protein levels of the mitotic inhibitor Wee1 kinase and the corresponding attenuation in Cdk1 activity is responsible for the rescuing effect of dnt1Δ on SIN mutants. Finally, our data also suggest that Dnt1 modulates Wee1 activity in parallel with SCF-mediated Wee1 degradation. Therefore, this study reveals an unexpected missing link between the nucleolar protein Dnt1 and the SIN signaling pathway, which is mediated by the Cdk1 regulator Wee1 kinase. Our findings also define a novel mode of regulation of Wee1 and Cdk1, which is important for integration of the signals controlling the SIN pathway in fission yeast.

  2. The microtubule destabilizing protein stathmin controls the transition from dividing neuronal precursors to postmitotic neurons during adult hippocampal neurogenesis.

    PubMed

    Boekhoorn, Karin; van Dis, Vera; Goedknegt, Erika; Sobel, André; Lucassen, Paul J; Hoogenraad, Casper C

    2014-12-01

    The hippocampus is one of the two areas in the mammalian brain where adult neurogenesis occurs. Adult neurogenesis is well known to be involved in hippocampal physiological functions as well as pathophysiological conditions. Microtubules (MTs), providing intracellular transport, stability, and transmitting force, are indispensable for neurogenesis by facilitating cell division, migration, growth, and differentiation. Although there are several examples of MT-stabilizing proteins regulating different aspects of adult neurogenesis, relatively little is known about the function of MT-destabilizing proteins. Stathmin is such a MT-destabilizing protein largely restricted to the CNS, and in contrast to its developmental family members, stathmin is also expressed at significant levels in the adult brain, notably in areas involved in adult neurogenesis. Here, we show an important role for stathmin during adult neurogenesis in the subgranular zone of the mouse hippocampus. After carefully mapping stathmin expression in the adult dentate gyrus (DG), we investigated its role in hippocampal neurogenesis making use of stathmin knockout mice. Although hippocampus development appears normal in these animals, different aspects of adult neurogenesis are affected. First, the number of proliferating Ki-67+ cells is decreased in stathmin knockout mice, as well as the expression of the immature markers Nestin and PSA-NCAM. However, newborn cells that do survive express more frequently the adult marker NeuN and have a more mature morphology. Furthermore, our data suggest that migration in the DG might be affected. We propose a model in which stathmin controls the transition from neuronal precursors to early postmitotic neurons. PMID:24909416

  3. 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

  4. 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.

  5. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

    PubMed Central

    Diao, Jiajie; Grob, Patricia; Cipriano, Daniel J; Kyoung, Minjoung; Zhang, Yunxiang; Shah, Sachi; Nguyen, Amie; Padolina, Mark; Srivastava, Ankita; Vrljic, Marija; Shah, Ankita; Nogales, Eva; Chu, Steven; Brunger, Axel T

    2012-01-01

    The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways. DOI: http://dx.doi.org/10.7554/eLife.00109.001 PMID:23240085

  6. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

    PubMed

    Diao, Jiajie; Grob, Patricia; Cipriano, Daniel J; Kyoung, Minjoung; Zhang, Yunxiang; Shah, Sachi; Nguyen, Amie; Padolina, Mark; Srivastava, Ankita; Vrljic, Marija; Shah, Ankita; Nogales, Eva; Chu, Steven; Brunger, Axel T

    2012-12-13

    The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.

  7. 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).

  8. Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone.

    PubMed

    Jonsson, Nina; Sävneby, Anna; Gullberg, Maria; Evertsson, Kim; Klingel, Karin; Lindberg, A Michael

    2015-06-01

    Recombination is an important feature in the evolution of the Enterovirus genus. Phylogenetic studies of enteroviruses have revealed that the capsid genomic region (P1) is type specific, while the parts of the genome coding for the non-structural proteins (P2-P3) are species specific. Hence, the genome may be regarded as consisting of two modules that evolve independently. In this study, it was investigated whether the non-structural coding part of the genome in one type could support replication of a virus with a P1 region from another type of the same species. A cassette vector (pCas) containing a full-length cDNA copy of coxsackievirus B5 (CVB5) was used as a replicative backbone. The P1 region of pCas was replaced with the corresponding part from coxsackievirus B3 Nancy (CVB3N), coxsackievirus B6 Schmitt (CVB6S), and echovirus 7 Wallace (E7W), all members of the Enterovirus B species. The replication efficiency after transfection with clone-derived in vitro transcribed RNA was studied and compared with that of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values, tissue culture infectivity dose 50 %, and plaque-forming unit titers comparable to viruses generated from the pCas construct. In addition to this, a clone without the P1 region was also constructed, and Western Blot and immunofluorescence staining analysis showed that the viral genome could be translated and replicated despite the lack of the structural protein-coding region. To conclude, the replicative backbone of the CVB5 cassette vector supports replication of intraspecies constructs with P1 regions derived from other members of the Enterovirus B species. In addition to this, the replicative backbone can be both translated and replicated without the presence of a P1 region.

  9. (1)H, (13)C and (15)N backbone resonance assignments and dynamic properties of the PDZ tandem of Whirlin.

    PubMed

    Delhommel, Florent; Wolff, Nicolas; Cordier, Florence

    2016-10-01

    Mammals perceive sounds thanks to mechanosensory hair cells located in the inner ear. The stereocilia of these cells are tightly bound together in bundles by a network of cadherins and scaffolding proteins. Stereocilia deflection induces stretching of this network and is responsible for hair cell depolarization that triggers the neuronal message, transducing the mechanical signal into an electric signal transmissible to the brain. Nearly all proteins involved in this mechano-electrical transduction network contain short C-terminal motifs of interaction with PDZ domains (PSD-95, Discs Large, ZO-1). Interestingly only two of these proteins encompass PDZ domains: Harmonin and Whirlin. As our first step towards a comprehensive structural study of Whirlin, we have assigned the (1)H, (13)C and (15)N backbone resonances of a tandem formed by the first two PDZ domains of Whirlin, reported the secondary structure elements of this tandem as predicted by the TALOS+ server and evaluated its dynamics from (15)N relaxation measurements.

  10. Data Acquisition Backbone Core DABC release v1.0

    NASA Astrophysics Data System (ADS)

    Adamczewski-Musch, J.; Essel, H. G.; Kurz, N.; Linev, S.

    2010-04-01

    The Data Acquisition Backbone Core (DABC) is a general purpose software framework designed for the implementation of a wide-range of data acquisition systems - from various small detector test beds to high performance systems. DABC consists of a compact data-flow kernel and a number of plug-ins for various functional components like data inputs, device drivers, user functional modules and applications. DABC provides configurable components for implementing event building over fast networks like InfiniBand or Gigabit Ethernet. A generic Java GUI provides the dynamic control and visualization of control parameters and commands, provided by DIM servers. A first set of application plug-ins has been implemented to use DABC as event builder for the front-end components of the GSI standard DAQ system MBS (Multi Branch System). Another application covers the connection to DAQ readout chains from detector front-end boards (N-XYTER) linked to read-out controller boards (ROC) over UDP into DABC for event building, archiving and data serving. This was applied for data taking in the September 2008 test beamtime for the CBM experiment at GSI. DABC version 1.0 is released and available from the website.

  11. 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

  12. 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.

  13. 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.

  14. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition.

    PubMed

    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching

    2013-01-01

    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  15. 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.

  16. Dynamic anchoring transitions at aqueous-liquid crystal interfaces induced by specific and non-specific binding of vesicles to proteins.

    PubMed

    Tan, Lie Na; Abbott, Nicholas L

    2015-07-01

    This paper reports on the dynamics of continuous anchoring transitions at interfaces formed between nematic liquid crystals (LCs, 4'-pentyl-4-cyanobiphenyl (5CB)) and immiscible aqueous phases that are induced by either non-specific or specific interactions between phospholipid vesicles and proteins adsorbed at the LC interfaces. By analyzing the dynamic response of LCs to non-specific adsorption of lipids onto bovine serum albumin (BSA)-decorated LC interfaces, we provide evidence that the LC anchoring transitions are slower than diffusion-controlled accumulation of lipid at the interface, consistent with the hypothesis that the LC transition involves lateral reorganization of proteins and lipids at the interface. Significantly, optical measurements of the tilt angle of the LC as a function of the amount of lipid captured at the interface were found to be quantitatively consistent with theoretical predictions of LC anchoring directed by nanoscopic domains of molecules that cause planar (protein) and homeotropic (lipid) anchoring of the LC. Finally, specific binding interactions between the antibody-decorated LC interfaces and vesicles (through antibody-antigen recognition) greatly accelerated the continuous LC anchoring transitions, with dynamics that were measured to scale with the logarithm of the ligand composition of the vesicles (over four orders of magnitude). The latter dynamics were found to be strongly influenced by addition of synthetic surfactants, consistent with our proposal that the rate-limiting step underlying the response of the LC was the transfer of lipids from captured vesicles into the protein-decorated LC interface. Overall, the results presented in this paper provide quantitative insight into the origin of continuous anchoring transitions triggered by vesicles at protein-decorated LC interfaces and, more broadly, guidance for the design of stimuli-responsive LC systems.

  17. 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.

  18. 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

  19. 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

    2016-04-28

    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.

  20. Phase behaviors, molecular and supramolecular structures in polymers containing rigid-rod backbones with cyanobiphenyl side chains

    NASA Astrophysics Data System (ADS)

    Ruan, Jrjeng

    One of the most important and challenging topics in materials chemistry involves designing nano-structures in synthetic materials via self-assembly for various highly technical applications. A specially designed combined liquid crystalline polymer containing a polyester backbone with cyanobiphenyl side chains has been studied in aspects of phase behaviors and crystal structures. The triclinic crystal phases identified in this series of polymer are all found to be constricted by 4-monomer unit cells. This discovery of 4-monomer triclinic unit cells motivates a search for the existence of supramolecular phases and understanding the possible molecular packing. A series of newly designed polyimides, which are composed of aromatic polyimide backbones with 4-cyanobiphenyl mesogens on the side chains has been synthesized. This series of polymers possesses a lesser degree of coupling between the backbones and side chains, which indicates the possibility of microphase separation between them. The representative polyimides of BPDA-7CBBP and BPDA-11CBBP in this series, in which 4-cyanobiphenyl side chains are connected onto the backbones through seven and eleven methylene units respectively have systematically studied in this research. Two crystal forms were recognized in BPDA-11CBBA, and one of them possesses six repeating units in one monoclinic unit cell. Moreover, the existence of a supramolecular phase has been proposed based on 2D WAXD fiber patterns. In the case of BPDA-7CBBP, three crystal forms were identified: two of them are constructed by triclinic lattices with large unit cells. The numbers of repeating units in those unit cells are seven and eight, respectively. Complicated phase behaviors including a second-order transition between the supramolecular phase and a high-order liquid crystal phase have been explored. The fact that large unit cells in both polymers with the numbers of repeating units in unit cells being 6, 7, and 8 leads to an important issue for

  1. 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

  2. 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

  3. The Light-Harvesting Chlorophyll a/b Binding Proteins Lhcb1 and Lhcb2 Play Complementary Roles during State Transitions in Arabidopsis[C][W][OPEN

    PubMed Central

    Pietrzykowska, Malgorzata; Suorsa, Marjaana; Semchonok, Dmitry A.; Tikkanen, Mikko; Boekema, Egbert J.; Aro, Eva-Mari

    2014-01-01

    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

  4. Ruthenium-catalyzed olefin metathesis accelerated by the steric effect of the backbone substituent in cyclic (alkyl)(amino) carbenes.

    PubMed

    Zhang, Jun; Song, Shangfei; Wang, Xiao; Jiao, Jiajun; Shi, Min

    2013-10-21

    Three ruthenium complexes bearing backbone-monosubstituted CAACs were prepared and displayed dramatic improvement in catalytic efficiency not only in RCM reaction but also in the ethenolysis of methyl oleate, compared to those bearing backbone-disubstituted CAACs. PMID:24013192

  5. Ruthenium-catalyzed olefin metathesis accelerated by the steric effect of the backbone substituent in cyclic (alkyl)(amino) carbenes.

    PubMed

    Zhang, Jun; Song, Shangfei; Wang, Xiao; Jiao, Jiajun; Shi, Min

    2013-10-21

    Three ruthenium complexes bearing backbone-monosubstituted CAACs were prepared and displayed dramatic improvement in catalytic efficiency not only in RCM reaction but also in the ethenolysis of methyl oleate, compared to those bearing backbone-disubstituted CAACs.

  6. Hair-cycle-dependent expression of parathyroid hormone-related protein and its type I receptor: evidence for regulation at the anagen to catagen transition.

    PubMed

    Cho, Yong Mee; Woodard, Grant L; Dunbar, Maureen; Gocken, Todd; Jimènez, Juan A; Foley, John

    2003-05-01

    The humoral hypercalcemia factor parathyroid hormone-related protein is a paracrine-signaling molecule that regulates the development of several organ systems, including the skin. In pathologic circumstances such as hypercalcemia and in development, parathyroid hormone-related protein signaling appears to be mediated by the type I parathyroid hormone/parathyroid hormone-related protein receptor. In order to clarify the role of the ligand and receptor pair in cutaneous biology, gene expression was monitored in a series of murine skin samples ranging from embryonic day 14 to 2 y with in situ hybridization and RNase protection. In all samples, high levels of parathyroid hormone-related protein transcripts were exclusively expressed in the developing and adult hair follicle but were not observed in the interfollicular epidermis. In the adult, parathyroid hormone-related protein mRNA expression was dynamically regulated as a function of the murine hair cycle in a way similar to other signaling molecules that regulate the anagen to catagen transition. PTH receptor transcripts were abundantly expressed in the developing dermis. In the adult skin, PTH receptor mRNA was markedly reduced, but again demonstrated hair-cycle-dependent expression. The dorsal skin of the keratin 14-parathyroid hormone-related protein mouse was used to evaluate the impact of overexpression of the peptide on the murine hair cycle. All types of hair were 30-40% shorter in adult keratin 14-parathyroid hormone-related protein mice as compared with wild-type littermates. This appeared to result from a premature entry into the catagen phase of the hair cycle. Finally, the relationship between parathyroid hormone-related protein signaling and other growth factors that regulate the hair cycle was examined by cross-breeding experiments employing keratin 14-parathyroid hormone-related protein mice and fibroblast growth factor-5-knockout mice. It appears that parathyroid hormone-related protein and

  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. Attosecond Electron Delocalization in the Conduction Band through the Phosphate Backbone of Genomic DNA

    NASA Astrophysics Data System (ADS)

    Ikeura-Sekiguchi, Hiromi; Sekiguchi, Tetsuhiro

    2007-11-01

    Partial density of states in the empty conduction band of the phosphate backbone sites in DNA was probed using energy-dependent resonant Auger spectroscopy. Results show that genomic DNA with periodic backbones exhibits an extended state despite separation of each phosphate group by an insulating sugar group. In antisense DNA with an aperiodic backbone, the equivalent state is localized. Remarkably rapid electron delocalization occurs at ca. 740 attoseconds for wet DNA, as estimated using the core-hole clock method. Such delocalization is comparable to the Fermi velocity of carbon nanotubes.

  9. High-risk human papillomavirus infections and overexpression of p53 protein as prognostic indicators in transitional cell carcinoma of the urinary bladder.

    PubMed

    Furihata, M; Inoue, K; Ohtsuki, Y; Hashimoto, H; Terao, N; Fujita, Y

    1993-10-15

    Ninety Japanese patients with transitional cell carcinoma of the urinary bladder were investigated for tumor incorporation of DNA for high-risk human papillomavirus (HPV) types 16, 18, and 33 by in situ hybridization with biotinylated DNA probes. In addition, immunohistochemical analysis of p53 protein expression was performed with an antibody to p53 protein. Twenty-eight tumors were positive for HPV DNA, and multiple HPV infection was detected in 17 cases. Positive nuclear staining of cancer cells by the antibody to p53 protein was detected in 32 cases. DNA for HPV 16, 18, and/or 33 and the overexpression of p53 protein were simultaneously observed in 6 tumors by using a mirror section method. The overexpression of p53 protein was frequently detected in invasive and nonpapillary tumors (P < 0.05) and in high grade tumors (P < 0.05). In contrast, HPV infection was more common in noninvasive and papillary tumors (P < 0.01). The patients with tumors positive for HPV DNA and/or p53 antibody had a significantly worse survival rate (P < 0.05). These results suggest that HPV infection or overexpression of p53 protein may be related to tumor behavior and may indicate a relatively poor prognosis in patients with transitional cell carcinoma.

  10. 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

  11. 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

  12. 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

  13. Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR.

    PubMed

    Knight, Michael J; Pell, Andrew J; Bertini, Ivano; Felli, Isabella C; Gonnelli, Leonardo; Pierattelli, Roberta; Herrmann, Torsten; Emsley, Lyndon; Pintacuda, Guido

    2012-07-10

    We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

  14. 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

  15. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds

    NASA Astrophysics Data System (ADS)

    Cino, Elio A.; Soares, Iaci N.; Pedrote, Murilo M.; de Oliveira, Guilherme A. P.; Silva, Jerson L.

    2016-09-01

    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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. In a changing environment, network backbone upgrades emerge as a wise investment.

    PubMed

    Cupito, M C

    1997-05-01

    The numbers, locations and needs of users change constantly, but they'll always want more bandwidth. Many experts say that upgrading to higher-speed backbones seems to be the smart investment for unsettled times.

  2. Determination of peptide backbone torsion angles using double-quantum dipolar recoupling solid-state NMR spectroscopy.

    PubMed

    Mehta, Manish A; Eddy, Matthew T; McNeill, Seth A; Mills, Frank D; Long, Joanna R

    2008-02-20

    Several approaches for utilizing dipolar recoupling solid-state NMR (ssNMR) techniques to determine local structure at high resolution in peptides and proteins have been developed. However, many of these techniques measure only one torsion angle or are accurate for only certain classes of secondary structure. Additionally, the efficiency with which these dipolar recoupling experiments suppress the deleterious effects of chemical shift anisotropy (CSA) at high magnetic field strengths varies. Dipolar recoupling with a windowless sequence (DRAWS) has proven to be an effective pulse sequence for exciting double-quantum (DQ) coherences between adjacent carbonyl carbons along the peptide backbone. By allowing this DQ coherence to evolve, it is possible to measure the relative orientations of the CSA tensors and subsequently use this information to determine the Ramachandran torsion angles phi and psi. Here, we explore the accuracies of the assumptions made in interpreting DQ-DRAWS data and demonstrate their fidelity in measuring torsion angles corresponding to a variety of secondary structures irrespective of hydrogen-bonding patterns. It is shown how a simple choice of isotopic labels and experimental conditions allows accurate measurement of backbone secondary structures without any prior knowledge. This approach is considerably more sensitive for determining structure in helices and has comparable accuracy for beta-sheet and extended conformations relative to other methods. We also illustrate the ability of DQ-DRAWS to distinguish between structures in heterogeneous samples.

  3. 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

  4. Transport properties of a single-molecular diode with one backbone, and two backbones in parallel: Frontier orbital analysis and NEGF-DFT study

    NASA Astrophysics Data System (ADS)

    Zahedi, Ehsan

    2015-05-01

    The conductance and electronic transport properties of a single-molecular diode with one backbone ( 1), and two backbones in parallel ( 2) have been investigated using frontier orbital analysis, and the NEGF formalism combined with DFT. The frontier orbital analysis results demonstrate that the electron transport from one end of the studied molecules to other end is symmetrically allowed and the conductance of the molecule with two parallel backbones is more than the molecule with a single backbone. Transmission spectra study based on the NEGF-DFT of the selected molecules sandwiched between two gold (1 1 1) electrodes showed that, due to a higher coupling between the two electrodes and the molecule 2, the zero-bias conductance is more than twice that of the other molecular junction. Transmission spectra under different biases showed that the maximum constructive interference exists at the bias voltage 0.2, while in some of the biases destructive effects are observed. I- V curves showed that the rectifying directions of molecular junctions 1 and 2 are opposite.

  5. Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase.

    PubMed

    Cao, Shufen; Mao, Xi'an; Liu, Deli; Buck, Matthias

    2013-10-01

    Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein-protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295-304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many (1)H(N), (13)C, and (15)N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N-H correlations in the (1)H-(15)N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77-83 and residues 127-131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1

  6. Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase.

    PubMed

    Cao, Shufen; Mao, Xi'an; Liu, Deli; Buck, Matthias

    2013-10-01

    Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein-protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295-304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many (1)H(N), (13)C, and (15)N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N-H correlations in the (1)H-(15)N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77-83 and residues 127-131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. [Protein conformational dynamics of crambin in crystal, solution and in the trajectories of molecular dynamics simulations].

    PubMed

    Abaturov, L V; Nosova, N G

    2013-01-01

    Atomic displacement parameters--B factors of the eight crambin crystal structures obtained at 0.54-1.5 angstroms resolution and temperatures of 100-293K have been analyzed. The comparable contributions to the B factor values are the intramolecular motions which are modeled by the harmonic vibration calculations and derived from the molecular dynamics simulation (MD) as well as rigid body changes in the position of a protein molecule as a whole. In solution for the average NMR structure of crambin the amplitudes of the backbone atomic fluctuations of the most residues of the segments with the regular backbone conformations are close to the amplitudes of the small scale harmonic vibrations. For the same residues the probability of the medium scale fluctuations fixed by the hydrogen exchange method is very low. The restricted conformational mobility of those segments is coupled with the depressed amplitudes of the fluctuation changes of the tertiary structure registered by the residue accessibility changes in an ensemble of NMR structures that forms the average NMR structure of crambin. The amplitudes of temperature fluctuations of backbone atoms and the tertiary structure raise in the segment with the irregular conformations, turn and loops. In the same segments the amplitudes of the calculated harmonic vibrations also increase, but to a lesser extent and especially in the interhelical loop with the most strong and complicated fluctuation changes of the backbone conformation. In solution for the NMR structure in this loop the conformational transitions occur between the conformational substates separated by the energy barriers, but they are not observed even in the long 100 ns trajectories from the MD simulation of crambin. These strong local fluctuation changes of the structure may play a key role in the protein functioning and modern performance improvements in the MD simulation techniques are oriented to increase the probability of protein appearance in the

  12. Sequence-specific 1H, 13C and 15N 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

  13. 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.

  14. 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.

  15. 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.

  16. Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing.

    PubMed

    Bejerman, Nicolás; Mann, Krin S; Dietzgen, Ralf G

    2016-09-15

    Plants employ RNA silencing as an innate defense mechanism against viruses. As a counter-defense, plant viruses have evolved to express RNA silencing suppressor proteins (RSS), which target one or more steps of the silencing pathway. In this study, we show that the phosphoprotein (P) encoded by the negative-sense RNA virus alfalfa dwarf virus (ADV), a species of the genus Cytorhabdovirus, family Rhabdoviridae, is a suppressor of RNA silencing. ADV P has a relatively weak local RSS activity, and does not prevent siRNA accumulation. On the other hand, ADV P strongly suppresses systemic RNA silencing, but does not interfere with the short-distance spread of silencing, which is consistent with its lack of inhibition of siRNA accumulation. The mechanism of suppression appears to involve ADV P binding to RNA-induced silencing complex proteins AGO1 and AGO4 as shown in protein-protein interaction assays when ectopically expressed. In planta, we demonstrate that ADV P likely functions by inhibiting miRNA-guided AGO1 cleavage and prevents transitive amplification by repressing the production of secondary siRNAs. As recently described for lettuce necrotic yellows cytorhabdovirus P, but in contrast to other viral RSS known to disrupt AGO activity, ADV P sequence does not contain any recognizable GW/WG or F-box motifs, which suggests that cytorhabdovirus P proteins may use alternative motifs to bind to AGO proteins.

  17. 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

  18. A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L.).

    PubMed

    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

  19. Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing.

    PubMed

    Bejerman, Nicolás; Mann, Krin S; Dietzgen, Ralf G

    2016-09-15

    Plants employ RNA silencing as an innate defense mechanism against viruses. As a counter-defense, plant viruses have evolved to express RNA silencing suppressor proteins (RSS), which target one or more steps of the silencing pathway. In this study, we show that the phosphoprotein (P) encoded by the negative-sense RNA virus alfalfa dwarf virus (ADV), a species of the genus Cytorhabdovirus, family Rhabdoviridae, is a suppressor of RNA silencing. ADV P has a relatively weak local RSS activity, and does not prevent siRNA accumulation. On the other hand, ADV P strongly suppresses systemic RNA silencing, but does not interfere with the short-distance spread of silencing, which is consistent with its lack of inhibition of siRNA accumulation. The mechanism of suppression appears to involve ADV P binding to RNA-induced silencing complex proteins AGO1 and AGO4 as shown in protein-protein interaction assays when ectopically expressed. In planta, we demonstrate that ADV P likely functions by inhibiting miRNA-guided AGO1 cleavage and prevents transitive amplification by repressing the production of secondary siRNAs. As recently described for lettuce necrotic yellows cytorhabdovirus P, but in contrast to other viral RSS known to disrupt AGO activity, ADV P sequence does not contain any recognizable GW/WG or F-box motifs, which suggests that cytorhabdovirus P proteins may use alternative motifs to bind to AGO proteins. PMID:27543392

  20. Evidence of coexistence of change of caged dynamics at T(g) and the dynamic transition at T(d) in solvated proteins.

    PubMed

    Capaccioli, S; Ngai, K L; Ancherbak, S; Paciaroni, A

    2012-02-16

    Mössbauer spectroscopy and neutron scattering measurements on proteins embedded in solvents including water and aqueous mixtures have emphasized the observation of the distinctive temperature dependence of the atomic mean square displacements, , commonly referred to as the dynamic transition at some temperature T(d). At low temperatures, increases slowly, but it assumes stronger temperature dependence after crossing T(d), which depends on the time/frequency resolution of the spectrometer. Various authors have made connection of the dynamics of solvated proteins, including the dynamic transition to that of glass-forming substances. Notwithstanding, no connection is made to the similar change of temperature dependence of obtained by quasielastic neutron scattering when crossing the glass transition temperature T(g), generally observed in inorganic, organic, and polymeric glass-formers. Evidences are presented here to show that such a change of the temperature dependence of from neutron scattering at T(g) is present in hydrated or solvated proteins, as well as in the solvent used, unsurprisingly since the latter is just another organic glass-former. If unaware of the existence of such a crossover of at T(g), and if present, it can be mistaken as the dynamic transition at T(d) with the ill consequences of underestimating T(d) by the lower value T(g) and confusing the identification of the origin of the dynamic transition. The obtained by neutron scattering at not so low temperatures has contributions from the dissipation of molecules while caged by the anharmonic intermolecular potential at times before dissolution of cages by the onset of the Johari-Goldstein β-relaxation or of the merged α-β relaxation. The universal change of at T(g) of glass-formers, independent of the spectrometer resolution, had been rationalized by sensitivity to change in volume and entropy of the dissipation of the caged molecules and its

  1. Whether proton transition to the triphosphate tail of ATP occurs at protein kinase environment: A Car-Parrinello ab initio molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Yang, Sheng-Yong; Zou, Jun; Xiang, Ming-Li; Xie, Guo-Bin; Shi, Bing; Wei, Yu-Quan

    Protonation state of the triphosphate tail of ATP (adenosine triphosphate) in protein environment is a fundamental issue, which has significant impact on the mechanism investigation of biochemical processes with ATP involved. Proton transition from surroundings (water molecule coordinating to magnesium, HW; amino group of Lys, HL) to the ATP tail in the catalytic core of protein kinase found recently disproved the commonly accepted deprotonation state of ATP tail. In this account, Car-Parrinello ab initio molecular dynamics (CP-AIMD) method has been employed to examine whether the proton transition occurs. To provide a comparison basis for the dynamics simulations, static quantum mechanics (QM), and combined quantum mechanics and molecular mechanics (QM/MM) calculations have also been carried out. Consistent results have been obtained that complete transition of hydrogen from the surroundings to the triphosphate tail of ATP is not allowed. The most dominant conformations correspond to the ones with HW bonding to O(W) and H-bonding to O(ATP), [O(W)-HW···O(ATP)], HL bonding to N(Lys) and H-bonding to O(ATP), [N(Lys)-HL···O(ATP)]. Metastable structures with one hydrogen atom bonding with two heavy atoms (hydrogen acceptors) were also located by our dynamic simulations. This bonding mode can satisfy the hungering for hydrogen of the two heavy atoms simultaneously.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. Interactions of TRIS [tris(hydroxymethyl)aminomethane] and related buffers with peptide backbone: thermodynamic characterization.

    PubMed

    Taha, Mohamed; Lee, Ming-Jer

    2010-10-21

    In a situation which is far from ideal, many buffers have been found to be quite reactive, besides maintaining their stable pH values. On the basis of apparent transfer free energies (ΔG(tr)'), through solubility measurements the interactions of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly(2)), triglycine (Gly(3)), and tetraglycine (Gly(4)), with several common neutral pH, amine-based buffers have been studied. The biological buffers studied in this work, including TRIS, TES, TAPS, TAPSO, and TABS are structurally related and all contain TRIS groups. These buffers have pK(a) values ranging from 7.5-9.0, which allow them to be used in biological, biochemical or environmental studies. We observed negative values of ΔG(tr)' for Gly(3) and Gly(4) from water to buffer, indicating that the interactions are favorable. However, the ΔG(tr)' values are positive for Gly and Gly(2), revealing unfavorable interactions, which except for the latter in TRIS buffer are negative. The surprising result in our data is the unexpected extraordinarily high favorable interactions between TRIS buffer and peptides (in comparison with the effect of the most common denaturants, urea and guanidine hydrochloride). The transfer free energies (ΔG(tr)') of the peptide backbone unit (-CH(2)C=O-NH-) contributions have been estimated from ΔG(tr)' values. We have also investigated the interactions of TRIS buffer with Bovine Serum Albumin (BSA), as a globular protein, using dynamic light scattering (DLS), zeta potential, UV-Visible absorption, fluorescence and Raman spectroscopy measurements. The results indicated that TRIS buffer stabilized the BSA molecules.

  7. Conserved Patterns in Backbone Torsional Changes Allow For Single Base Flipping from Duplex DNA with Minimal Distortion of the Double Helix

    PubMed Central

    Banavali, Nilesh K.; Huang, Niu; MacKerell, Alexander D.

    2008-01-01

    Base flipping is a structural mechanism common to many DNA processing and repair enzymes. Changes in the local backbone torsions that occur during base flipping and the effect of environment on their behavior are of particular interest in understanding different base flipping mechanisms. In the present study, structures sampled during umbrella sampling molecular dynamics (MD) simulations of base flipping in aqueous and protein-bound environments, carried out with two different MD simulation strategies, are analyzed to find the most significant phosphodiester backbone distortions in the vicinity of the flipping base. Torsional sampling on the 5′ side of the flipping base during flipping through the major groove show similarities to the torsional sampling on the 3′ side during flipping through the minor groove and vice versa. In differing environments, this behavior varies only marginally. These compensating torsional changes in the DNA backbone on 5′ and 3′ sides of the flipping base limit overall distortion of the DNA double helix during single base flipping. Rotameric intermediate states observed during base flipping are identified and postulated to be metastable states implicated in both large-scale structural changes and functional effects of chemical modifications in DNA. PMID:16771353

  8. 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

  9. 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

  10. 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-de