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Sample records for flexible protein loops

  1. Efficient Algorithms to Explore Conformation Spaces of Flexible Protein Loops

    PubMed Central

    Yao, Peggy; Dhanik, Ankur; Marz, Nathan; Propper, Ryan; Kou, Charles; Liu, Guanfeng; van den Bedem, Henry; Latombe, Jean-Claude; Halperin-Landsberg, Inbal; Altman, Russ Biagio

    2009-01-01

    Several applications in biology—e.g., incorporation of protein flexibility in ligand docking algorithms, interpretation of fuzzy X-ray crystallographic data, and homology modeling—require computing the internal parameters of a flexible fragment (usually, a loop) of a protein in order to connect its termini to the rest of the protein without causing any steric clash inside the loop and with the rest of the protein. One must often sample many such conformations in order to explore and adequately represent the conformational range of the studied loop. While sampling must be fast, it is made difficult by the fact that two conflicting constraints—kinematic closure and clash avoidance—must be satisfied concurrently. This paper describes two efficient and complementary sampling algorithms to explore the space of closed clash-free conformations of a flexible protein loop. The “seed sampling” algorithm samples broadly from this space, while the “deformation sampling” algorithm uses seed conformations as starting points to explore the conformation space around them at a finer grain. Computational results are presented for various loops ranging from 5 to 25 residues. More specific results also show that the combination of the sampling algorithms with a functional site prediction software (FEATURE) makes it possible to compute and recognize calcium-binding loop conformations. The sampling algorithms are implemented in a toolkit, called LoopTK, which is available at https://simtk.org/home/looptk. PMID:18989041

  2. Efficient algorithms to explore conformation spaces of flexible protein loops.

    PubMed

    Yao, Peggy; Dhanik, Ankur; Marz, Nathan; Propper, Ryan; Kou, Charles; Liu, Guanfeng; van den Bedem, Henry; Latombe, Jean-Claude; Halperin-Landsberg, Inbal; Altman, Russ Biagio

    2008-01-01

    Several applications in biology - e.g., incorporation of protein flexibility in ligand docking algorithms, interpretation of fuzzy X-ray crystallographic data, and homology modeling - require computing the internal parameters of a flexible fragment (usually, a loop) of a protein in order to connect its termini to the rest of the protein without causing any steric clash. One must often sample many such conformations in order to explore and adequately represent the conformational range of the studied loop. While sampling must be fast, it is made difficult by the fact that two conflicting constraints - kinematic closure and clash avoidance - must be satisfied concurrently. This paper describes two efficient and complementary sampling algorithms to explore the space of closed clash-free conformations of a flexible protein loop. The "seed sampling" algorithm samples broadly from this space, while the "deformation sampling" algorithm uses seed conformations as starting points to explore the conformation space around them at a finer grain. Computational results are presented for various loops ranging from 5 to 25 residues. More specific results also show that the combination of the sampling algorithms with a functional site prediction software (FEATURE) makes it possible to compute and recognize calcium-binding loop conformations. The sampling algorithms are implemented in a toolkit (LoopTK), which is available at https://simtk.org/home/looptk.

  3. Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

    PubMed Central

    2015-01-01

    GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL. PMID:25127419

  4. Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

    PubMed Central

    Rau, Michael; Stump, W. Tom; Hall, Kathleen B.

    2012-01-01

    Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops. PMID:23012481

  5. Structure of the Neisserial Outer Membrane Protein Opa60: Loop Flexibility Essential to Receptor Recognition and Bacterial Engulfment

    PubMed Central

    2015-01-01

    The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors. PMID:24813921

  6. Deployable radiator with flexible line loop

    NASA Technical Reports Server (NTRS)

    Keeler, Bryan V. (Inventor); Lehtinen, Arthur Mathias (Inventor); McGee, Billy W. (Inventor)

    2003-01-01

    Radiator assembly (10) for use on a spacecraft (12) is provided including at least one radiator panel assembly (26) repeatably movable between a panel stowed position (28) and a panel deployed position (36), at least two flexible lines (40) in fluid communication with the at least one radiator panel assembly (26) and repeatably movable between a stowage loop (42) and a flattened deployed loop (44).

  7. Leaping of a flexible loop on water

    NASA Astrophysics Data System (ADS)

    Kim, Ho-Young; Yang, Eun Jin; Lee, Min-Hee; Shin, Bongsu

    2008-11-01

    Small aquatic arthropods, such as water striders and fishing spiders, are able to leap on water to a height several times their body length. We study a simple model using a floating flexible loop to provide fundamental understanding and mimicking principle of the leaping on water. Motion of a loop, initially bent into an ellipse from equilibrium circular shape using a thin thread, is visualized with a high speed camera upon cutting the thread with a laser. We find that the loop may merely oscillate while afloat, penetrate into the water, or soar into air depending on the hydrophobicity, the bending stiffness, the weight and the degree of initial deflection of the loop. We also construct a scaling law for the leaping height by balancing the initial elastic bending energy with the loop's translational and vibrational energy and a loss imparted to the water in the forms of interfacial, kinetic and viscous energy.

  8. Experimental and Theoretical Study of the Movement of the Wpd Flexible Loop of Human Protein Tyrosine Phosphatase PTP1B in Complex with Halide Ions

    NASA Astrophysics Data System (ADS)

    Katz, Aline; Saenz-Méndez, Patricia; Cousido-Siah, Alexandra; Podjarny, Alberto D.; Ventura, Oscar N.

    2012-11-01

    Protein tyrosine phosphorylation is a post-translational modification mechanism, crucial for the regulation of nearly all aspects of cell life. This dynamic, reversible process is regulated by the balanced opposing activity of protein tyrosine kinases and protein tyrosine phosphatases. In particular, the protein tyrosine phosphatase 1B (PTP1B) is implicated in the regulation of the insulin-receptor activity, leptin-stimulated signal transduction pathways and other clinically relevant metabolic routes, and it has been found overexpressed or overregulated in human breasts, colon and ovary cancers. The WPD loop of the enzyme presents an inherent flexibility, and it plays a fundamental role in the enzymatic catalysis, turning it into a potential target in the design of new efficient PTP1B inhibitors. In order to determine the interactions that control the spatial conformation adopted by the WPD loop, complexes between the enzyme and halide ions (Br- and I- in particular) were crystallized and their crystallographic structure determined, and the collective movements of the aforementioned complexes were studied through Molecular Dynamics (MD) simulations. Both studies yielded concordant results, indicating the existence of a relationship between the identity of the ion present in the complex and the strength of the interactions it establishes with the surrounding protein residues.

  9. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

    PubMed

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B P; Davison, Paul A; Brindley, Amanda A; Hunter, C Neil; Guallar, Victor; Sobotka, Roman

    2015-11-20

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

  10. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway*

    PubMed Central

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B. P.; Davison, Paul A.; Brindley, Amanda A.; Hunter, C. Neil; Guallar, Victor; Sobotka, Roman

    2015-01-01

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway. PMID:26446792

  11. Protein flexibility as a biosignal.

    PubMed

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  12. Dynamics of closed-loop systems containing flexible bodies

    NASA Technical Reports Server (NTRS)

    Tadikonda, Sivakumar S. K.; Singh, Ramendra P.

    1991-01-01

    An important characteristic of flexible multibody systems containing closed-loop topologies is that the component modes used to describe individual bodies will no longer be independent because of loop closure constraints. Thus, the issue of component modal selection becomes even more complicated. In addition, the foreshortening effect that has been studied extensively in the literature in the context of open-loop topologies will also be present in these constraint equations. Simulation results presented demonstrate the effects of modal selection and foreshortening on the dynamic response of closed-loop flexible systems.

  13. Dynamics of closed-loop systems containing flexible bodies

    NASA Technical Reports Server (NTRS)

    Tadikonda, Sivakumar S. K.; Singh, Ramendra P.

    1991-01-01

    An important characteristic of flexible multibody systems containing closed-loop topologies is that the component modes used to describe individual bodies will no longer be independent because of loop closure constraints. Thus, the issue of component modal selection becomes even more complicated. In addition, the foreshortening effect that has been studied extensively in the literature in the context of open-loop topologies will also be present in these constraint equations. Simulation results presented demonstrate the effects of modal selection and foreshortening on the dynamic response of closed-loop flexible systems.

  14. Fast loop modeling for protein structures

    NASA Astrophysics Data System (ADS)

    Zhang, Jiong; Nguyen, Son; Shang, Yi; Xu, Dong; Kosztin, Ioan

    2015-03-01

    X-ray crystallography is the main method for determining 3D protein structures. In many cases, however, flexible loop regions of proteins cannot be resolved by this approach. This leads to incomplete structures in the protein data bank, preventing further computational study and analysis of these proteins. For instance, all-atom molecular dynamics (MD) simulation studies of structure-function relationship require complete protein structures. To address this shortcoming, we have developed and implemented an efficient computational method for building missing protein loops. The method is database driven and uses deep learning and multi-dimensional scaling algorithms. We have implemented the method as a simple stand-alone program, which can also be used as a plugin in existing molecular modeling software, e.g., VMD. The quality and stability of the generated structures are assessed and tested via energy scoring functions and by equilibrium MD simulations. The proposed method can also be used in template-based protein structure prediction. Work supported by the National Institutes of Health [R01 GM100701]. Computer time was provided by the University of Missouri Bioinformatics Consortium.

  15. Two tandem flexible loops in a viscous flow

    NASA Astrophysics Data System (ADS)

    Ye, Huilin; Wei, Heng; Huang, Haibo; Lu, Xi-yun

    2017-02-01

    Interaction between two tandem flexible loops with tension and bending stiffness in a viscous flow is investigated by numerical simulations. In most cases, the heads of the loops facing the oncoming flow are fixed but flapping around the head is allowed. The effect of the gap distance between the two passive flapping loops (G) on the drag coefficient is investigated in detail. Here, for the first time, the sudden drag force reduction at a specific G, i.e., Gc for the downstream loop has been found in the two tandem flexible loops system. It is different from the drag "jump" behavior in the two tandem rigid cylinder system. Although the drag is partially associated with the flapping amplitude, the drag force reduction of the downstream loop may be mainly attributed to flow regimes transition or vortices merging mode transition. The vortices merging is also analysed from a Lagrangian viewpoint, which gives insight into the mechanism. The effects of Reynolds number (20 ≤ Re ≤100 ) , bending coefficient (10-4≤K ≤2 ×10-2 ) , and tension coefficient (10 ≤S ≤1000 ) are also investigated and the relevant mechanism is explored. If the head of the downstream loop is set free to move laterally, the critical distance (Gc) where the sudden drag reduction occurs would further decrease compared to the fixed case, which is due to the early shedding regime transition.

  16. ModLoop: automated modeling of loops in protein structures.

    PubMed

    Fiser, András; Sali, Andrej

    2003-12-12

    ModLoop is a web server for automated modeling of loops in protein structures. The input is the atomic coordinates of the protein structure in the Protein Data Bank format, and the specification of the starting and ending residues of one or more segments to be modeled, containing no more than 20 residues in total. The output is the coordinates of the non-hydrogen atoms in the modeled segments. A user provides the input to the server via a simple web interface, and receives the output by e-mail. The server relies on the loop modeling routine in MODELLER that predicts the loop conformations by satisfaction of spatial restraints, without relying on a database of known protein structures. For a rapid response, ModLoop runs on a cluster of Linux PC computers. The server is freely accessible to academic users at http://salilab.org/modloop

  17. Protein short loop prediction in terms of a structural alphabet.

    PubMed

    Tyagi, Manoj; Bornot, Aurélie; Offmann, Bernard; de Brevern, Alexandre G

    2009-08-01

    Loops connect regular secondary structures. In many instances, they are known to play crucial biological roles. To bypass the limitation of secondary structure description, we previously defined a structural alphabet composed of 16 structural prototypes, called Protein Blocks (PBs). It leads to an accurate description of every region of 3D protein backbones and has been used in local structure prediction. In the present study, we used our structural alphabet to predict the loops connecting two repetitive structures. Thus, we showed interest to take into account the flanking regions, leading to prediction rate improvement up to 19.8%, but we also underline the sensitivity of such an approach. This research can be used to propose different structures for the loops and to probe and sample their flexibility. It is a useful tool for ab initio loop prediction and leads to insights into flexible docking approach.

  18. Unbiased, scalable sampling of protein loop conformations from probabilistic priors

    PubMed Central

    2013-01-01

    Background Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Results Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Conclusion Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion. PMID:24565175

  19. Flexible Proteins at the Origin of Life

    PubMed Central

    Pohorille, Andrew; Wilson, Michael A.; Shannon, Gareth

    2017-01-01

    Almost all modern proteins possess well-defined, relatively rigid scaffolds that provide structural preorganization for desired functions. Such scaffolds require the sufficient length of a polypeptide chain and extensive evolutionary optimization. How ancestral proteins attained functionality, even though they were most likely markedly smaller than their contemporary descendants, remains a major, unresolved question in the origin of life. On the basis of evidence from experiments and computer simulations, we argue that at least some of the earliest water-soluble and membrane proteins were markedly more flexible than their modern counterparts. As an example, we consider a small, evolved in vitro ligase, based on a novel architecture that may be the archetype of primordial enzymes. The protein does not contain a hydrophobic core or conventional elements of the secondary structure characteristic of modern water-soluble proteins, but instead is built of a flexible, catalytic loop supported by a small hydrophilic core containing zinc atoms. It appears that disorder in the polypeptide chain imparts robustness to mutations in the protein core. Simple ion channels, likely the earliest membrane protein assemblies, could also be quite flexible, but still retain their functionality, again in contrast to their modern descendants. This is demonstrated in the example of antiamoebin, which can serve as a useful model of small peptides forming ancestral ion channels. Common features of the earliest, functional protein architectures discussed here include not only their flexibility, but also a low level of evolutionary optimization and heterogeneity in amino acid composition and, possibly, the type of peptide bonds in the protein backbone. PMID:28587235

  20. Flexible Proteins at the Origin of Life.

    PubMed

    Pohorille, Andrew; Wilson, Michael A; Shannon, Gareth

    2017-06-05

    Almost all modern proteins possess well-defined, relatively rigid scaffolds that provide structural preorganization for desired functions. Such scaffolds require the sufficient length of a polypeptide chain and extensive evolutionary optimization. How ancestral proteins attained functionality, even though they were most likely markedly smaller than their contemporary descendants, remains a major, unresolved question in the origin of life. On the basis of evidence from experiments and computer simulations, we argue that at least some of the earliest water-soluble and membrane proteins were markedly more flexible than their modern counterparts. As an example, we consider a small, evolved in vitro ligase, based on a novel architecture that may be the archetype of primordial enzymes. The protein does not contain a hydrophobic core or conventional elements of the secondary structure characteristic of modern water-soluble proteins, but instead is built of a flexible, catalytic loop supported by a small hydrophilic core containing zinc atoms. It appears that disorder in the polypeptide chain imparts robustness to mutations in the protein core. Simple ion channels, likely the earliest membrane protein assemblies, could also be quite flexible, but still retain their functionality, again in contrast to their modern descendants. This is demonstrated in the example of antiamoebin, which can serve as a useful model of small peptides forming ancestral ion channels. Common features of the earliest, functional protein architectures discussed here include not only their flexibility, but also a low level of evolutionary optimization and heterogeneity in amino acid composition and, possibly, the type of peptide bonds in the protein backbone.

  1. Propylene loop heat pipe with a lightweight, flexible, deployable radiator

    NASA Astrophysics Data System (ADS)

    Krotiuk, William J.; Crowley, Christopher J.; Rozzi, Jay C.

    2002-01-01

    This paper describes the design and fabrication of a propylene Loop Heat Pipe (LHP) with a flexible, deployable radiator intended to provide a lightweight spacecraft thermal management system. The paper presents the results of testing the LHP in an environmental chamber and in a thermal-vacuum chamber. Observations regarding operational and startup characteristics of the LHP are provided, and a comparison of design calculations to test results is presented. .

  2. Two strategies to engineer flexible loops for improved enzyme thermostability

    NASA Astrophysics Data System (ADS)

    Yu, Haoran; Yan, Yihan; Zhang, Cheng; Dalby, Paul A.

    2017-02-01

    Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes.

  3. Two strategies to engineer flexible loops for improved enzyme thermostability

    PubMed Central

    Yu, Haoran; Yan, Yihan; Zhang, Cheng; Dalby, Paul A.

    2017-01-01

    Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes. PMID:28145457

  4. Two strategies to engineer flexible loops for improved enzyme thermostability.

    PubMed

    Yu, Haoran; Yan, Yihan; Zhang, Cheng; Dalby, Paul A

    2017-02-01

    Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a "back to consensus mutations" approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes.

  5. Crystal packing effects on protein loops.

    PubMed

    Rapp, Chaya S; Pollack, Rena M

    2005-07-01

    The effects of crystal packing on protein loop structures are examined by (1) a comparison of loops in proteins that have been crystallized in alternate packing arrangements, and (2) theoretical prediction of loops both with and without the inclusion of the crystal environment. Results show that in a minority of cases, loop geometries are dependent on crystal packing effects. Explicit representation of the crystal environment in a loop prediction algorithm can be used to model these effects and to reconstruct the structures, and relative energies, of a loop in alternative packing environments. By comparing prediction results with and without the inclusion of the crystal environment, the loop prediction algorithm can further be used to identify cases in which a crystal structure does not represent the most stable state of a loop in solution. We anticipate that this capability has implications for structural biology.

  6. The importance of slow motions for protein functional loops.

    PubMed

    Skliros, Aris; Zimmermann, Michael T; Chakraborty, Debkanta; Saraswathi, Saras; Katebi, Ataur R; Leelananda, Sumudu P; Kloczkowski, Andrzej; Jernigan, Robert L

    2012-02-07

    Loops in proteins that connect secondary structures such as alpha-helix and beta-sheet, are often on the surface and may play a critical role in some functions of a protein. The mobility of loops is central for the motional freedom and flexibility requirements of active-site loops and may play a critical role for some functions. The structures and behaviors of loops have not been studied much in the context of the whole structure and its overall motions, especially how these might be coupled. Here we investigate loop motions by using coarse-grained structures (C(α) atoms only) to solve the motions of the system by applying Lagrange equations with elastic network models to learn about which loops move in an independent fashion and which move in coordination with domain motions, faster and slower, respectively. The normal modes of the system are calculated using eigen-decomposition of the stiffness matrix. The contribution of individual modes and groups of modes is investigated for their effects on all residues in each loop by using Fourier analyses. Our results indicate overall that the motions of functional sets of loops behave in similar ways as the whole structure. But overall only a relatively few loops move in coordination with the dominant slow modes of motion, and these are often closely related to function.

  7. Statistical database analysis of the role of loop dynamics for protein-protein complex formation and allostery.

    PubMed

    Gu, Yina; Li, Da-Wei; Brüschweiler, Rafael

    2017-06-15

    Protein loops show rich conformational dynamics properties on a wide range of timescales as they play an essential role for many cellular functions during protein-protein interactions and recognition processes. However, little is known about the detail behavior of loops upon protein binding including allostery. We report the loop motions and their dominant timescales for a library of 230 proteins that form protein-protein complexes using the ToeLoop predictor of loop dynamics. We applied the analysis to proteins in both their complex and free state and relate specific loop properties to their role in protein recognition. We observe a strong tendency of loops that move on relatively slow timescales of tens of ns to sub-μs to be directly involved in binding and recognition processes. Complex formation leads to a significant reduction in loop flexibility at the binding interface, but in a number of cases it can also trigger increased flexibility in distal loops in response to allosteric conformational changes. The importance of loop dynamics and allostery is highlighted by a case study of an antibody-antigen complex. Furthermore, we explored the relationship between loop dynamics and experimental binding affinities and found that a prevalence of high loop rigidity at the binding interface is an indicator of increased binding strength. http://spin.ccic.ohio-state.edu/index.php/toeloopppi. bruschweiler.1@osu.edu. Supplementary data are available at Bioinformatics online.

  8. Conformational selection of protein kinase A revealed by flexible-ligand flexible-protein docking.

    PubMed

    Huang, Zunnan; Wong, Chung F

    2009-03-01

    Protein kinases have high structural plasticity: their structure can change significantly, depending on what ligands are bound to them. Rigid-protein docking methods are not capable of describing such effects. Here, we present a new flexible-ligand flexible-protein docking model in which the protein can adopt conformations between two extremes observed experimentally. The model utilized a molecular dynamics-based simulated annealing cycling protocol and a distance-dependent dielectric model to perform docking. By testing this model on docking four diverse ligands to protein kinase A, we found that the ligands were able to dock successfully to the protein with the proper conformations of the protein induced. By imposing relatively soft conformational restraints to the protein during docking, this model reduced computational costs yet permitted essential conformational changes that were essential for these inhibitors to dock properly to the protein. For example, without adequate movement of the glycine-rich loop, it was difficult for the ligands to move from the surface of the protein to the binding site. In addition, these simulations called for better ways to compare simulation results with experiment other than using the popular root-mean-square deviation between the structure of a ligand in a docking pose and that in experiment because the structure of the protein also changed. In this work, we also calculated the correlation coefficient between protein-ligand/protein-protein distances in the docking structure and those in the crystal structure to check how well a ligand docked into the binding site of the protein and whether the proper conformation of the protein was induced.

  9. Stochastic model for protein flexibility analysis.

    PubMed

    Xia, Kelin; Wei, Guo-Wei

    2013-12-01

    Protein flexibility is an intrinsic property and plays a fundamental role in protein functions. Computational analysis of protein flexibility is crucial to protein function prediction, macromolecular flexible docking, and rational drug design. Most current approaches for protein flexibility analysis are based on Hamiltonian mechanics. We introduce a stochastic model to study protein flexibility. The essential idea is to analyze the free induction decay of a perturbed protein structural probability, which satisfies the master equation. The transition probability matrix is constructed by using probability density estimators including monotonically decreasing radial basis functions. We show that the proposed stochastic model gives rise to some of the best predictions of Debye-Waller factors or B factors for three sets of protein data introduced in the literature.

  10. Proteins mediating DNA loops effectively block transcription.

    PubMed

    Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

    2017-07-01

    Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  11. HMG Proteins and DNA Flexibility in Transcription Activation

    PubMed Central

    Ross, Eric D.; Hardwidge, Philip R.; Maher, L. James

    2001-01-01

    The relative stiffness of naked DNA is evident from measured values of longitudinal persistence length (∼150 bp) and torsional persistence length (∼180 bp). These parameters predict that certain arrangements of eukaryotic transcription activator proteins in gene promoters should be much more effective than others in fostering protein-protein interactions with the basal RNA polymerase II transcription apparatus. Thus, if such interactions require some kind of DNA looping, DNA loop energies should depend sensitively on helical phasing of protein binding sites, loop size, and intrinsic DNA curvature within the loop. Using families of artificial transcription templates where these parameters were varied, we were surprised to find that the degree of transcription activation by arrays of Gal4-VP1 transcription activators in HeLa cell nuclear extract was sensitive only to the linear distance separating a basal promoter from an array of bound activators on DNA templates. We now examine the hypothesis that this unexpected result is due to factors in the extract that act to enhance apparent DNA flexibility. We demonstrate that HeLa cell nuclear extract is rich in a heat-resistant activity that dramatically enhances apparent DNA longitudinal and torsional flexibility. Recombinant mammalian high-mobility group 2 (HMG-2) protein can substitute for this activity. We propose that the abundance of HMG proteins in eukaryotic nuclei provides an environment in which DNA is made sufficiently flexible to remove many constraints on protein binding site arrangements that would otherwise limit efficient transcription activation to certain promoter geometries. PMID:11533247

  12. A Self-Organizing Algorithm for Modeling Protein Loops

    PubMed Central

    Liu, Pu; Zhu, Fangqiang; Rassokhin, Dmitrii N.; Agrafiotis, Dimitris K.

    2009-01-01

    Protein loops, the flexible short segments connecting two stable secondary structural units in proteins, play a critical role in protein structure and function. Constructing chemically sensible conformations of protein loops that seamlessly bridge the gap between the anchor points without introducing any steric collisions remains an open challenge. A variety of algorithms have been developed to tackle the loop closure problem, ranging from inverse kinematics to knowledge-based approaches that utilize pre-existing fragments extracted from known protein structures. However, many of these approaches focus on the generation of conformations that mainly satisfy the fixed end point condition, leaving the steric constraints to be resolved in subsequent post-processing steps. In the present work, we describe a simple solution that simultaneously satisfies not only the end point and steric conditions, but also chirality and planarity constraints. Starting from random initial atomic coordinates, each individual conformation is generated independently by using a simple alternating scheme of pairwise distance adjustments of randomly chosen atoms, followed by fast geometric matching of the conformationally rigid components of the constituent amino acids. The method is conceptually simple, numerically stable and computationally efficient. Very importantly, additional constraints, such as those derived from NMR experiments, hydrogen bonds or salt bridges, can be incorporated into the algorithm in a straightforward and inexpensive way, making the method ideal for solving more complex multi-loop problems. The remarkable performance and robustness of the algorithm are demonstrated on a set of protein loops of length 4, 8, and 12 that have been used in previous studies. PMID:19696883

  13. Disparate Degrees of Hypervariable Loop Flexibility Control T-Cell Receptor Cross-Reactivity, Specificity, and Binding Mechanism

    SciTech Connect

    Scott, Daniel R.; Borbulevych, Oleg Y.; Piepenbrink, Kurt H.; Corcelli, Steven A.; Baker, Brian M.

    2012-06-19

    {alpha}{beta} T-cell receptors (TCRs) recognize multiple antigenic peptides bound and presented by major histocompatibility complex molecules. TCR cross-reactivity has been attributed in part to the flexibility of TCR complementarity-determining region (CDR) loops, yet there have been limited direct studies of loop dynamics to determine the extent of its role. Here we studied the flexibility of the binding loops of the {alpha}{beta} TCR A6 using crystallographic, spectroscopic, and computational methods. A significant role for flexibility in binding and cross-reactivity was indicated only for the CDR3{alpha} and CDR3{beta} hypervariable loops. Examination of the energy landscapes of these two loops indicated that CDR3{beta} possesses a broad, smooth energy landscape, leading to rapid sampling in the free TCR of a range of conformations compatible with different ligands. The landscape for CDR3{alpha} is more rugged, resulting in more limited conformational sampling that leads to specificity for a reduced set of peptides as well as the major histocompatibility complex protein. In addition to informing on the mechanisms of cross-reactivity and specificity, the energy landscapes of the two loops indicate a complex mechanism for TCR binding, incorporating elements of both conformational selection and induced fit in a manner that blends features of popular models for TCR recognition.

  14. Mutational Analysis of Sclerostin Shows Importance of the Flexible Loop and the Cystine-Knot for Wnt-Signaling Inhibition

    PubMed Central

    Boschert, Verena; van Dinther, Maarten; Weidauer, Stella; van Pee, Katharina; Muth, Eva-Maria; ten Dijke, Peter; Mueller, Thomas D.

    2013-01-01

    The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured β-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different. PMID:24312339

  15. Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

    PubMed

    Álvarez, Lucía; Lewis-Ballester, Ariel; Roitberg, Adrián; Estrin, Darío A; Yeh, Syun-Ru; Marti, Marcelo A; Capece, Luciana

    2016-05-17

    Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

  16. The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair

    PubMed Central

    Storici, Francesca; Henneke, Ghislaine; Ferrari, Elena; Gordenin, Dmitry A.; Hübscher, Ulrich; Resnick, Michael A.

    2002-01-01

    The conserved, structure-specific flap endonuclease FEN1 cleaves 5′ DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G2 arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5′ flaps, the loop mutants exhibited partial ability to cut 5′ flaps when an adjacent single nucleotide 3′ flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5′ flap and the catalytic site. PMID:12411510

  17. Poly(dA:dT)-rich DNAs are highly flexible in the context of DNA looping.

    PubMed

    Johnson, Stephanie; Chen, Yi-Ju; Phillips, Rob

    2013-01-01

    Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant

  18. Removing the invariant salt bridge of parvalbumin increases flexibility in the AB-loop structure.

    PubMed

    Hoh, François; Cavé, Adrien; Strub, Marie Paule; Banères, Jean Louis; Padilla, André

    2009-08-01

    Parvalbumins (PVs) are calcium-buffer proteins that belong to the EF-hand family. Their N-terminal domain consists of two antiparallel helices A and B that make up a flat hydrophobic surface that is associated with the opposite side of the CD and EF binding sites. A single conserved Arg75-Glu81 salt bridge is buried in this hydrophobic interface. The structure of a rat PV mutant in which Arg75 was replaced by alanine was solved by molecular replacement. Unexpectedly, a large distance deviation of 7.8 A was observed for the AB loop but not for the residues that flank the R75A mutation. The thermal stability of the calcium-loaded form is lower (T(m) = 352.0 K; DeltaT(m) = -11.4 K) than that of the wild-type protein and the apo mutant is unfolded at room temperature. Weaker calcium or magnesium affinities were also measured for the R75A mutant (Ca(2+): K(1) = 4.21 x 10(7) M(-1), K(2) = 6.18 x 10(6) M(-1); Mg(2+): K(1) = 2.98 x 10(4) M(-1), K(2) = 3.09 x 10(3) M(-1)). Finally, comparison of the B factors showed an increase in the flexibility of the AB loop that is consistent with this region being more exposed to solvent in the mutant. The mutant structure therefore demonstrates the role of the salt bridge in attaching the nonbinding AB domain to the remaining protein core. Normal-mode analysis indeed indicated an altered orientation of the AB domain with regard to the CD-EF binding domains.

  19. Looping charged elastic rods: applications to protein-induced DNA loop formation.

    PubMed

    Cherstvy, A G

    2011-01-01

    We analyze looping of thin charged elastic filaments under applied torques and end forces, using the solution of linear elasticity theory equations. In application to DNA, we account for its polyelectrolyte character and charge renormalization, calculating electrostatic energies stored in the loops. We argue that the standard theory of electrostatic persistence is only valid when the loop's radius of curvature and close-contact distance are much larger than the Debye screening length. We predict that larger twist rates are required to trigger looping of charged rods as compared with neutral ones. We then analyze loop shapes formed on charged filaments of finite length, mimicking DNA looping by proteins with two DNA-binding domains. We find optimal loop shapes at different salt amounts, minimizing the sum of DNA elastic, DNA electrostatic, and protein elastic energies. We implement a simple model where intercharge repulsions do not affect the loop shape directly but can choose the energy-optimized shape from the allowed loop types. At low salt concentrations more open loops are favored due to enhanced repulsion of DNA charges, consistent with the results of computer simulations on formation of DNA loops by lac repressor. Then, we model the precise geometry of DNA binding by the lac tetramer and explore loop shapes, varying the confined DNA length and protein opening angle. The characteristics of complexes obtained, such as the total loop energy, stretching forces required to maintain its shape, and the reduction of electrostatic energy with increment of salt, are in good agreement with the outcomes of more elaborate numerical calculations for lac-repressor-induced DNA looping.

  20. Closed loop control of a robot assisted smart flexible needle for percutaneous intervention.

    PubMed

    Maria Joseph, F O; Hutapea, P; Dicker, A; Yu, Y; Podder, T

    2015-08-01

    This paper presents the experimental evaluation of a coordinated control system for a robot and robot-driven shape memory alloy (SMA) actuated smart flexible needle capable of following a curved path for percutaneous intervention. The robot driving the needle is considered the outer loop and the non-linear SMA actuated flexible needle system comprises the inner loop. The two feedback control loops are coordinated in such a way that the robot drives the needle while monitoring the needle's actual deflection against a preplanned ideal trajectory, so that the needle tip reaches the target location within an acceptable accuracy. In air and in water experimental results are presented to validate the ability of the proposed coordinated controller to track the overall desired trajectory which includes the combined trajectory of the robot driver and the needle.

  1. A flexible loop as a functional element in the catalytic mechanism of nucleoside hydrolase from Trypanosoma vivax.

    PubMed

    Vandemeulebroucke, An; De Vos, Stefan; Van Holsbeke, Els; Steyaert, Jan; Versées, Wim

    2008-08-08

    The nucleoside hydrolase of Trypanosoma vivax hydrolyzes the N-glycosidic bond of purine nucleosides. Structural and kinetic studies on this enzyme have suggested a catalytic role for a flexible loop in the vicinity of the active sites. Here we present the analysis of the role of this flexible loop via the combination of a proline scan of the loop, loop deletion mutagenesis, steady state and pre-steady state analysis, and x-ray crystallography. Our analysis reveals that this loop has an important role in leaving group activation and product release. The catalytic role involves the entire loop and could only be perturbed by deletion of the entire loop and not by single site mutagenesis. We present evidence that the loop closes over the active site during catalysis, thereby ordering a water channel that is involved in leaving group activation. Once chemistry has taken place, the loop dynamics determine the rate of product release.

  2. Fast Protein Loop Sampling and Structure Prediction Using Distance-Guided Sequential Chain-Growth Monte Carlo Method

    PubMed Central

    Tang, Ke; Zhang, Jinfeng; Liang, Jie

    2014-01-01

    Loops in proteins are flexible regions connecting regular secondary structures. They are often involved in protein functions through interacting with other molecules. The irregularity and flexibility of loops make their structures difficult to determine experimentally and challenging to model computationally. Conformation sampling and energy evaluation are the two key components in loop modeling. We have developed a new method for loop conformation sampling and prediction based on a chain growth sequential Monte Carlo sampling strategy, called Distance-guided Sequential chain-Growth Monte Carlo (DiSGro). With an energy function designed specifically for loops, our method can efficiently generate high quality loop conformations with low energy that are enriched with near-native loop structures. The average minimum global backbone RMSD for 1,000 conformations of 12-residue loops is Å, with a lowest energy RMSD of Å, and an average ensemble RMSD of Å. A novel geometric criterion is applied to speed up calculations. The computational cost of generating 1,000 conformations for each of the x loops in a benchmark dataset is only about cpu minutes for 12-residue loops, compared to ca cpu minutes using the FALCm method. Test results on benchmark datasets show that DiSGro performs comparably or better than previous successful methods, while requiring far less computing time. DiSGro is especially effective in modeling longer loops (– residues). PMID:24763317

  3. Shortening a loop can increase protein native state entropy.

    PubMed

    Gavrilov, Yulian; Dagan, Shlomi; Levy, Yaakov

    2015-12-01

    Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. © 2015 Wiley Periodicals, Inc.

  4. Increased flexibility decreases antifreeze protein activity.

    PubMed

    Patel, Shruti N; Graether, Steffen P

    2010-12-01

    Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single α-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg37 HPLC6, nonamidated HPLC6 Ala37, amidated HPLC6 Ala37, and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still α-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface. Copyright © 2010 The Protein Society.

  5. The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.

    PubMed Central

    Okoniewska, M; Tanaka, T; Yada, R Y

    2000-01-01

    Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes. PMID:10861225

  6. Distributed flow sensing for closed-loop speed control of a flexible fish robot.

    PubMed

    Zhang, Feitian; Lagor, Francis D; Yeo, Derrick; Washington, Patrick; Paley, Derek A

    2015-10-23

    Flexibility plays an important role in fish behavior by enabling high maneuverability for predator avoidance and swimming in turbulent flow. This paper presents a novel flexible fish robot equipped with distributed pressure sensors for flow sensing. The body of the robot is molded from soft, hyperelastic material, which provides flexibility. Its Joukowski-foil shape is conducive to modeling the fluid analytically. A quasi-steady potential-flow model is adopted for real-time flow estimation, whereas a discrete-time vortex-shedding flow model is used for higher-fidelity simulation. The dynamics for the flexible fish robot yield a reduced model for one-dimensional swimming. A recursive Bayesian filter assimilates pressure measurements to estimate flow speed, angle of attack, and foil camber. The closed-loop speed-control strategy combines an inverse-mapping feedforward controller based on an average model derived for periodic actuation of angle-of-attack and a proportional-integral feedback controller utilizing the estimated flow information. Simulation and experimental results are presented to show the effectiveness of the estimation and control strategy. The paper provides a systematic approach to distributed flow sensing for closed-loop speed control of a flexible fish robot by regulating the flapping amplitude.

  7. The central loop of Escherichia coli glutamine synthetase is flexible and functionally passive.

    PubMed

    Pearson, Josh T; Dabrowski, Michael J; Kung, Irene; Atkins, William M

    2005-04-15

    Bacterial glutamine synthetases (GSs) are dodecameric aggregates comprised of two face-to-face hexameric rings, which form a cylindrical aqueous channel. Available crystal structures indicate that each subunit provides a 'central loop' that protrudes into this channel. Residues on either side of this loop contribute directly to substrate or metal ion cofactor binding. Although it has been suggested that this conspicuous structural feature may be functionally important, a systematic structure-function analysis of this loop has not been done. Here, we examine the behavior of a cysteine mutant, E165C, which yields inter-subunit disulfide bonds connecting the central loops. The inter-subunit disulfide bonds are readily detected by electrospray ionization mass spectrometry. Based on molecular models, the disulfide bonds would form only if the engineered cysteines on adjacent subunits moved approximately 5 A. Surprisingly, inter-subunit disulfide bonds between the central loops caused no detectable changes in the KMs for glutamate or ATP, nor the KD for either ATP or the transition state analog (L)-methionine sulfoximine (MSOX). Furthermore, covalent and quantitative adduction of the E165C mutant with iodo-acetamido-pyrene yielded nearly fully active enzyme bearing fluorescent pyrene excimers. The relative contribution of pyrene monomers to excimers in the steady state fluorescence is temperature dependent, suggesting thermal equilibrium between loop conformational states. However, the monomer-excimer ratio is independent of ligands such as MSOX, glutamate, or Mn2+. These results validate the suspected flexibility of the central loop, but raise significant doubt about its direct functional role in GS catalysis via conformational switching, including the proposed regulation of GS via ADP-ribosylation within this loop.

  8. Protein-protein docking with a reduced protein model accounting for side-chain flexibility.

    PubMed

    Zacharias, Martin

    2003-06-01

    A protein-protein docking approach has been developed based on a reduced protein representation with up to three pseudo atoms per amino acid residue. Docking is performed by energy minimization in rotational and translational degrees of freedom. The reduced protein representation allows an efficient search for docking minima on the protein surfaces within. During docking, an effective energy function between pseudo atoms has been used based on amino acid size and physico-chemical character. Energy minimization of protein test complexes in the reduced representation results in geometries close to experiment with backbone root mean square deviations (RMSDs) of approximately 1 to 3 A for the mobile protein partner from the experimental geometry. For most test cases, the energy-minimized experimental structure scores among the top five energy minima in systematic docking studies when using both partners in their bound conformations. To account for side-chain conformational changes in case of using unbound protein conformations, a multicopy approach has been used to select the most favorable side-chain conformation during the docking process. The multicopy approach significantly improves the docking performance, using unbound (apo) binding partners without a significant increase in computer time. For most docking test systems using unbound partners, and without accounting for any information about the known binding geometry, a solution within approximately 2 to 3.5 A RMSD of the full mobile partner from the experimental geometry was found among the 40 top-scoring complexes. The approach could be extended to include protein loop flexibility, and might also be useful for docking of modeled protein structures.

  9. Protein Loop Closure Using Orientational Restraints from NMR Data

    NASA Astrophysics Data System (ADS)

    Tripathy, Chittaranjan; Zeng, Jianyang; Zhou, Pei; Donald, Bruce Randall

    Protein loops often play important roles in biological functions such as binding, recognition, catalytic activities and allosteric regulation. Modeling loops that are biophysically sensible is crucial to determining the functional specificity of a protein. A variety of algorithms ranging from robotics-inspired inverse kinematics methods to fragmentbased homology modeling techniques have been developed to predict protein loops. However, determining the 3D structures of loops using global orientational restraints on internuclear vectors, such as those obtained from residual dipolar coupling (RDC) data in solution Nuclear Magnetic Resonance (NMR) spectroscopy, has not been well studied. In this paper, we present a novel algorithm that determines the protein loop conformations using a minimal amount of RDC data. Our algorithm exploits the interplay between the sphero-conics derived from RDCs and the protein kinematics, and formulates the loop structure determination problem as a system of low-degree polynomial equations that can be solved exactly and in closed form. The roots of these polynomial equations, which encode the candidate conformations, are searched systematically, using efficient and provable pruning strategies that triage the vast majority of conformations, to enumerate or prune all possible loop conformations consistent with the data. Our algorithm guarantees completeness by ensuring that a possible loop conformation consistent with the data is never missed. This data-driven algorithm provides a way to assess the structural quality from experimental data with minimal modeling assumptions. We applied our algorithm to compute the loops of human ubiquitin, the FF Domain 2 of human transcription elongation factor CA150 (FF2), the DNA damage inducible protein I (DinI) and the third IgG-binding domain of Protein G (GB3) from experimental RDC data. A comparison of our results versus those obtained by using traditional structure determination protocols on the

  10. Engineering proteins for thermostability through rigidifying flexible sites.

    PubMed

    Yu, Haoran; Huang, He

    2014-01-01

    Engineering proteins for thermostability is an exciting and challenging field since it is critical for broadening the industrial use of recombinant proteins. Thermostability of proteins arises from the simultaneous effect of several forces such as hydrophobic interactions, disulfide bonds, salt bridges and hydrogen bonds. All of these interactions lead to decreased flexibility of polypeptide chain. Structural studies of mesophilic and thermophilic proteins showed that the latter need more rigid structures to compensate for increased thermal fluctuations. Hence flexibility can be an indicator to pinpoint weak spots for enhancing thermostability of enzymes. A strategy has been proven effective in enhancing proteins' thermostability with two steps: predict flexible sites of proteins firstly and then rigidify these sites. We refer to this approach as rigidify flexible sites (RFS) and give an overview of such a method through summarizing the methods to predict flexibility of a protein, the methods to rigidify residues with high flexibility and successful cases regarding enhancing thermostability of proteins using RFS.

  11. Zinc ion coordination as a modulating factor of the ZnuA histidine-rich loop flexibility: A molecular modeling and fluorescence spectroscopy study

    SciTech Connect

    Castelli, Silvia; Stella, Lorenzo; Petrarca, Patrizia; Battistoni, Andrea; Desideri, Alessandro; Falconi, Mattia

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer Fluorescence data indicate that the His-loop of ZnuA interacts with Zn{sup +2} ions. Black-Right-Pointing-Pointer The ZnuA structural model proposed validates these spectroscopic findings. Black-Right-Pointing-Pointer It is proposed that a zinc loaded His-loop may facilitate the ZnuA-ZnuB recognition. -- Abstract: ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the ATP-binding cassette-type periplasmic Zn-binding proteins. The zinc transporter ZnuABC is composed by three proteins: ZnuB, the membrane permease, ZnuC, the ATPase component and ZnuA, the soluble periplasmic metal-binding protein which captures Zn and delivers it to ZnuB. The ZnuA protein contains a charged flexible loop, rich in histidines and acidic residues, showing significant species-specific differences. Various studies have established that this loop contributes to the formation of a secondary zinc binding site, which has been proposed to be important in the acquisition of periplasmic Zn for its delivery to ZnuB or for regulation of zinc uptake. Due to its high mobility the structure of the histidine-rich loop has never been solved by X-ray diffraction studies. In this paper, through a combined use of molecular modeling, mutagenesis and fluorescence spectroscopy, we confirm the presence of two zinc binding sites characterized by different affinities for the metal ion and show that the flexibility of the loop is modulated by the binding of the zinc ions to the protein. The data obtained by fluorescence spectroscopy have then be used to validate a 3D model including the unsolved histidine-rich loop.

  12. Incorporation of protein flexibility and conformational energy penalties in docking screens to improve ligand discovery.

    PubMed

    Fischer, Marcus; Coleman, Ryan G; Fraser, James S; Shoichet, Brian K

    2014-07-01

    Proteins fluctuate between alternative conformations, which presents a challenge for ligand discovery because such flexibility is difficult to treat computationally owing to problems with conformational sampling and energy weighting. Here we describe a flexible docking method that samples and weights protein conformations using experimentally derived conformations as a guide. The crystallographically refined occupancies of these conformations, which are observable in an apo receptor structure, define energy penalties for docking. In a large prospective library screen, we identified new ligands that target specific receptor conformations of a cavity in cytochrome c peroxidase, and we confirm both ligand pose and associated receptor conformation predictions by crystallography. The inclusion of receptor flexibility led to ligands with new chemotypes and physical properties. By exploiting experimental measures of loop and side-chain flexibility, this method can be extended to the discovery of new ligands for hundreds of targets in the Protein Data Bank for which similar experimental information is available.

  13. Incorporation of protein flexibility and conformational energy penalties in docking screens to improve ligand discovery

    NASA Astrophysics Data System (ADS)

    Fischer, Marcus; Coleman, Ryan G.; Fraser, James S.; Shoichet, Brian K.

    2014-07-01

    Proteins fluctuate between alternative conformations, which presents a challenge for ligand discovery because such flexibility is difficult to treat computationally owing to problems with conformational sampling and energy weighting. Here we describe a flexible docking method that samples and weights protein conformations using experimentally derived conformations as a guide. The crystallographically refined occupancies of these conformations, which are observable in an apo receptor structure, define energy penalties for docking. In a large prospective library screen, we identified new ligands that target specific receptor conformations of a cavity in cytochrome c peroxidase, and we confirm both ligand pose and associated receptor conformation predictions by crystallography. The inclusion of receptor flexibility led to ligands with new chemotypes and physical properties. By exploiting experimental measures of loop and side-chain flexibility, this method can be extended to the discovery of new ligands for hundreds of targets in the Protein Data Bank for which similar experimental information is available.

  14. Mining protein loops using a structural alphabet and statistical exceptionality

    PubMed Central

    2010-01-01

    Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a

  15. Exploiting protein intrinsic flexibility in drug design.

    PubMed

    Lukman, Suryani; Verma, Chandra S; Fuentes, Gloria

    2014-01-01

    Molecular recognition in biological systems relies on the existence of specific attractive interactions between two partner molecules. Structure-based drug design seeks to identify and optimize such interactions between ligands and their protein targets. The approach followed in medicinal chemistry follows a combination of careful analysis of structural data together with experimental and/or theoretical studies on the system. This chapter focuses on the fact that a protein is not fully characterized by a single structure, but by an ensemble of states, some of them represent "hidden conformations" with cryptic binding sites. We highlight case studies where both experimental and computational methods have been used to mutually drive each other in an attempt to improve the success of the drug design approaches.Advances in both experimental techniques and computational methods have greatly improved our physico-chemical understanding of the functional mechanisms in biomolecules and opened a debate about the interplay between molecular structure and biomolecular function. The beautiful static pictures of protein structures may have led to neglecting the intrinsic protein flexibility, however we are entering a new era where more sophisticated methods are used to exploit this ability of macromolecules, and this will definitely lead to the inclusion of the notion in the pharmaceutical field of drug design.

  16. A multiscale model for simulating binding kinetics of proteins with flexible linkers.

    PubMed

    Chen, Jiawen; Xie, Zhong-Ru; Wu, Yinghao

    2014-10-01

    The kinetics of protein interactions are essential determinants in many cellular processes such as signal transduction and transcriptional regulation. Many proteins involved in these functions contain intrinsic disordered regions. This makes conformational flexibility become an unneglectable factor when studying the binding kinetic of these proteins. Compared with the binding of rigid proteins that is limited by diffusions, the binding mechanisms of proteins with internal flexibility are much more complicated. Using a small protein that contains two domains and a connecting loop as a testing system, we developed a multiscale simulation framework to study the role of flexible linkers in regulating kinetics of protein binding. The association and dissociation processes were implemented by a coarse-grained Monte-Carlo algorithm, while the conformational changes of the flexible linker were captured from all-atom molecular dynamic simulations. Our simulations illustrated that the presence of the extended domain linker can enhance the rate of protein association. On the other hand, the full-length flexible molecule is more difficult to dissociate than its two rigid domains but much easier than the molecule with a rigid linker. Overall, our studies demonstrated that both kinetics and thermodynamics of protein binding are closely modulated by the dynamic features of linker regions.

  17. FlexSnap: Flexible Non-sequential Protein Structure Alignment

    NASA Astrophysics Data System (ADS)

    Salem, Saeed; Zaki, Mohammed J.; Bystroff, Chris

    Proteins have evolved subject to energetic selection pressure for stability and flexibility. Structural similarity between proteins which have gone through conformational changes can be captured effectively if flexibility is considered. Topologically unrelated proteins that preserve secondary structure packing interactions can be detected if both flexibility and sequence permutations are considered. We propose the FlexSnap algorithm for flexible non-topological protein structural alignment. The effectiveness of FlexSnap is demonstrated by measuring the agreement of its alignments with manually curated non-sequential structural alignments. FlexSnap showed competitive results against state-of-the-art algorithms, like DALI, SARF2, MultiProt, FlexProt, and FATCAT.

  18. The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket.

    PubMed

    Meyners, Christian; Krämer, Andreas; Yildiz, Özkan; Meyer-Almes, Franz-Josef

    2017-07-01

    The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Experimental Study of Flexible Plate Vibration Control by Using Two-Loop Sliding Mode Control Strategy

    NASA Astrophysics Data System (ADS)

    Yang, Jingyu; Lin, Jiahui; Liu, Yuejun; Yang, Kang; Zhou, Lanwei; Chen, Guoping

    2016-06-01

    It is well known that intelligent control theory has been used in many research fields, novel modeling method (DROMM) is used for flexible rectangular active vibration control, and then the validity of new model is confirmed by comparing finite element model with new model. In this paper, taking advantage of the dynamics of flexible rectangular plate, a two-loop sliding mode (TSM) MIMO approach is introduced for designing multiple-input multiple-output continuous vibration control system, which can overcome uncertainties, disturbances or unstable dynamics. An illustrative example is given in order to show the feasibility of the method. Numerical simulations and experiment confirm the effectiveness of the proposed TSM MIMO controller.

  20. Experimental Study of Flexible Plate Vibration Control by Using Two-Loop Sliding Mode Control Strategy

    NASA Astrophysics Data System (ADS)

    Yang, Jingyu; Lin, Jiahui; Liu, Yuejun; Yang, Kang; Zhou, Lanwei; Chen, Guoping

    2017-08-01

    It is well known that intelligent control theory has been used in many research fields, novel modeling method (DROMM) is used for flexible rectangular active vibration control, and then the validity of new model is confirmed by comparing finite element model with new model. In this paper, taking advantage of the dynamics of flexible rectangular plate, a two-loop sliding mode (TSM) MIMO approach is introduced for designing multiple-input multiple-output continuous vibration control system, which can overcome uncertainties, disturbances or unstable dynamics. An illustrative example is given in order to show the feasibility of the method. Numerical simulations and experiment confirm the effectiveness of the proposed TSM MIMO controller.

  1. Design and Measurements of a Hybrid RF-MEMS Reconfigurable Loop Antenna on a Flexible Substrate (PREPRINT)

    DTIC Science & Technology

    2010-06-01

    AFRL-RY-WP-TP-2010-1147 DESIGN AND MEASUREMENTS OF A HYBRID RF-MEMS RECONFIGURABLE LOOP ANTENNA ON A FLEXIBLE SUBSTRATE (PREPRINT) Nelson... Antenna on a Flexible Substrate Nelson Sepulveda(1), Dimitrios E. Anagnostou* (2), Rafael A. Rodriguez-Solis(1), and John L. Ebel(3) (1... antenna was designed, fabricated (on a flexible substrate) and measured. The design concepts and measurement results are presented and discussed

  2. Conformational sampling and structure prediction of multiple interacting loops in soluble and β-barrel membrane proteins using multi-loop distance-guided chain-growth Monte Carlo method

    PubMed Central

    Tang, Ke; Wong, Samuel W.K.; Liu, Jun S.; Zhang, Jinfeng; Liang, Jie

    2015-01-01

    Motivation: Loops in proteins are often involved in biochemical functions. Their irregularity and flexibility make experimental structure determination and computational modeling challenging. Most current loop modeling methods focus on modeling single loops. In protein structure prediction, multiple loops often need to be modeled simultaneously. As interactions among loops in spatial proximity can be rather complex, sampling the conformations of multiple interacting loops is a challenging task. Results: In this study, we report a new method called multi-loop Distance-guided Sequential chain-Growth Monte Carlo (M-DiSGro) for prediction of the conformations of multiple interacting loops in proteins. Our method achieves an average RMSD of 1.93 Å for lowest energy conformations of 36 pairs of interacting protein loops with the total length ranging from 12 to 24 residues. We further constructed a data set containing proteins with 2, 3 and 4 interacting loops. For the most challenging target proteins with four loops, the average RMSD of the lowest energy conformations is 2.35 Å. Our method is also tested for predicting multiple loops in β-barrel membrane proteins. For outer-membrane protein G, the lowest energy conformation has a RMSD of 2.62 Å for the three extracellular interacting loops with a total length of 34 residues (12, 12 and 10 residues in each loop). Availability and implementation: The software is freely available at: tanto.bioe.uic.edu/m-DiSGro. Contact: jinfeng@stat.fsu.edu or jliang@uic.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25861965

  3. Trains, tails and loops of partially adsorbed semi-flexible filaments.

    PubMed

    Welch, David; Lettinga, M P; Ripoll, Marisol; Dogic, Zvonimir; Vliegenthart, Gerard A

    2015-10-14

    Polymer adsorption is a fundamental problem in statistical mechanics that has direct relevance to diverse disciplines ranging from biological lubrication to stability of colloidal suspensions. We combine experiments with computer simulations to investigate depletion induced adsorption of semi-flexible polymers onto a hard-wall. Three dimensional filament configurations of partially adsorbed F-actin polymers are visualized with total internal reflection fluorescence microscopy. This information is used to determine the location of the adsorption/desorption transition and extract the statistics of trains, tails and loops of partially adsorbed filament configurations. In contrast to long flexible filaments which primarily desorb by the formation of loops, the desorption of stiff, finite-sized filaments is largely driven by fluctuating filament tails. Simulations quantitatively reproduce our experimental data and allow us to extract universal laws that explain scaling of the adsorption-desorption transition with relevant microscopic parameters. Our results demonstrate how the adhesion strength, filament stiffness, length, as well as the configurational space accessible to the desorbed filament can be used to design the characteristics of filament adsorption and thus engineer properties of composite biopolymeric materials.

  4. Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

    NASA Astrophysics Data System (ADS)

    Lia, Giuseppe; Bensimon, David; Croquette, Vincent; Allemand, Jean-Francois; Dunlap, David; Lewis, Dale E. A.; Adhya, Sankar; Finzi, Laura

    2003-09-01

    The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of 40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

  5. Flexibility and small pockets at protein-protein interfaces: New insights into druggability.

    PubMed

    Jubb, Harry; Blundell, Tom L; Ascher, David B

    2015-10-01

    The transient assembly of multiprotein complexes mediates many aspects of cell regulation and signalling in living organisms. Modulation of the formation of these complexes through targeting protein-protein interfaces can offer greater selectivity than the inhibition of protein kinases, proteases or other post-translational regulatory enzymes using substrate, co-factor or transition state mimetics. However, capitalising on protein-protein interaction interfaces as drug targets has been hindered by the nature of interfaces that tend to offer binding sites lacking the well-defined large cavities of classical drug targets. In this review we posit that interfaces formed by concerted folding and binding (disorder-to-order transitions on binding) of one partner and other examples of interfaces where a protein partner is bound through a continuous epitope from a surface-exposed helix, flexible loop or chain extension may be more tractable for the development of "orthosteric", competitive chemical modulators; these interfaces tend to offer small-volume but deep pockets and/or larger grooves that may be bound tightly by small chemical entities. We discuss examples of such protein-protein interaction interfaces for which successful chemical modulators are being developed.

  6. Characterization and Prediction of Protein Flexibility Based on Structural Alphabets

    PubMed Central

    Liu, Bin

    2016-01-01

    Motivation. To assist efforts in determining and exploring the functional properties of proteins, it is desirable to characterize and predict protein flexibilities. Results. In this study, the conformational entropy is used as an indicator of the protein flexibility. We first explore whether the conformational change can capture the protein flexibility. The well-defined decoy structures are converted into one-dimensional series of letters from a structural alphabet. Four different structure alphabets, including the secondary structure in 3-class and 8-class, the PB structure alphabet (16-letter), and the DW structure alphabet (28-letter), are investigated. The conformational entropy is then calculated from the structure alphabet letters. Some of the proteins show high correlation between the conformation entropy and the protein flexibility. We then predict the protein flexibility from basic amino acid sequence. The local structures are predicted by the dual-layer model and the conformational entropy of the predicted class distribution is then calculated. The results show that the conformational entropy is a good indicator of the protein flexibility, but false positives remain a problem. The DW structure alphabet performs the best, which means that more subtle local structures can be captured by large number of structure alphabet letters. Overall this study provides a simple and efficient method for the characterization and prediction of the protein flexibility. PMID:27660756

  7. Systematic identification of protein combinations mediating chromatin looping

    PubMed Central

    Zhang, Kai; Li, Nan; Ainsworth, Richard I.; Wang, Wei

    2016-01-01

    Chromatin looping plays a pivotal role in gene expression and other biological processes through bringing distal regulatory elements into spatial proximity. The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in three-dimensional (3D) space. Previously, identification of DBP cooperation has been limited to those binding to neighbouring regions in the proximal linear genome (1D cooperation). Here we present the first study that integrates protein ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs. We develop a new network model that allows identification of cooperation between multiple DBPs and reveals cell-type-specific and -independent regulations. Using this framework, we retrieve many known and previously unknown 3D-cooperations between DBPs in chromosomal loops that may be a key factor in influencing the 3D organization of chromatin. PMID:27461729

  8. DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation.

    PubMed

    Boedicker, James Q; Garcia, Hernan G; Johnson, Stephanie; Phillips, Rob

    2013-12-01

    As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.

  9. DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

    NASA Astrophysics Data System (ADS)

    Boedicker, James Q.; Garcia, Hernan G.; Johnson, Stephanie; Phillips, Rob

    2013-12-01

    As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.

  10. Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization.

    PubMed

    Kolawole, Abimbola O; Li, Ming; Xia, Chunsheng; Fischer, Audrey E; Giacobbi, Nicholas S; Rippinger, Christine M; Proescher, Jody B G; Wu, Susan K; Bessling, Seneca L; Gamez, Monica; Yu, Chenchen; Zhang, Rebecca; Mehoke, Thomas S; Pipas, James M; Wolfe, Joshua T; Lin, Jeffrey S; Feldman, Andrew B; Smith, Thomas J; Wobus, Christiane E

    2014-04-01

    New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A'B' and E'F' loops. Six mutations in the E'F' loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the

  11. Identifying Functional Requirements for Flexible Airspace Management Concept Using Human-In-The-Loop Simulations

    NASA Technical Reports Server (NTRS)

    Lee, Paul U.; Bender, Kim; Pagan, Danielle

    2011-01-01

    Flexible Airspace Management (FAM) is a mid- term Next Generation Air Transportation System (NextGen) concept that allows dynamic changes to airspace configurations to meet the changes in the traffic demand. A series of human-in-the-loop (HITL) studies have identified procedures and decision support requirements needed to implement FAM. This paper outlines a suggested FAM procedure and associated decision support functionality based on these HITL studies. A description of both the tools used to support the HITLs and the planned NextGen technologies available in the mid-term are presented and compared. The mid-term implementation of several NextGen capabilities, specifically, upgrades to the Traffic Management Unit (TMU), the initial release of an en route automation system, the deployment of a digital data communication system, a more flexible voice communications network, and the introduction of a tool envisioned to manage and coordinate networked ground systems can support the implementation of the FAM concept. Because of the variability in the overall deployment schedule of the mid-term NextGen capabilities, the dependency of the individual NextGen capabilities are examined to determine their impact on a mid-term implementation of FAM. A cursory review of the different technologies suggests that new functionality slated for the new en route automation system is a critical enabling technology for FAM, as well as the functionality to manage and coordinate networked ground systems. Upgrades to the TMU are less critical but important nonetheless for FAM to be fully realized. Flexible voice communications network and digital data communication system could allow more flexible FAM operations but they are not as essential.

  12. Gaussian-weighted RMSD superposition of proteins: a structural comparison for flexible proteins and predicted protein structures.

    PubMed

    Damm, Kelly L; Carlson, Heather A

    2006-06-15

    Many proteins contain flexible structures such as loops and hinged domains. A simple root mean square deviation (RMSD) alignment of two different conformations of the same protein can be skewed by the difference between the mobile regions. To overcome this problem, we have developed a novel method to overlay two protein conformations by their atomic coordinates using a Gaussian-weighted RMSD (wRMSD) fit. The algorithm is based on the Kabsch least-squares method and determines an optimal transformation between two molecules by calculating the minimal weighted deviation between the two coordinate sets. Unlike other techniques that choose subsets of residues to overlay, all atoms are included in the wRMSD overlay. Atoms that barely move between the two conformations will have a greater weighting than those that have a large displacement. Our superposition tool has produced successful alignments when applied to proteins for which two conformations are known. The transformation calculation is heavily weighted by the coordinates of the static region of the two conformations, highlighting the range of flexibility in the overlaid structures. Lastly, we show how wRMSD fits can be used to evaluate predicted protein structures. Comparing a predicted fold to its experimentally determined target structure is another case of comparing two protein conformations of the same sequence, and the degree of alignment directly reflects the quality of the prediction.

  13. Efficient Computation of Closed-loop Frequency Response for Large Order Flexible Systems

    NASA Technical Reports Server (NTRS)

    Maghami, Peiman G.; Giesy, Daniel P.

    1997-01-01

    An efficient and robust computational scheme is given for the calculation of the frequency response function of a large order, flexible system implemented with a linear, time invariant control system. Advantage is taken of the highly structured sparsity of the system matrix of the plant based on a model of the structure using normal mode coordinates. The computational time per frequency point of the new computational scheme is a linear function of system size, a significant improvement over traditional, full-matrix techniques whose computational times per frequency point range from quadratic to cubic functions of system size. This permits the practical frequency domain analysis of systems of much larger order than by traditional, full-matrix techniques. Formulations are given for both open and closed loop loop systems. Numerical examples are presented showing the advantages of the present formulation over traditional approaches, both in speed and in accuracy. Using a model with 703 structural modes, a speed-up of almost two orders of magnitude was observed while accuracy improved by up to 5 decimal places.

  14. Contrasting HIV phylogenetic relationships and V3 loop protein similarities

    SciTech Connect

    Korber, B. Santa Fe Inst., NM ); Myers, G. )

    1992-01-01

    At least five distinct sequence subtypes of HIV-I can be identified from the major centers of the AMS pandemic. While it is too early to tell whether these subtypes are serologically or phenotypically similar or distinct in terms of properties such as pathogenicity and transmissibility, we can begin to investigate their potential for phenotypic divergence at the protein sequence level. Phylogenetic analysis of HIV DNA sequences is being widely used to examine lineages of different viral strains as they evolve and spread throughout the globe. We have identified five distinct HIV-1 subtypes (designated A-E), or clades, based on phylogenetic clustering patterns generated from genetic information from both the gag and envelope (env) genes from a spectrum of international isolates. Our initial observations concerning both HIV-1 and HIV-2 sequences indicate that conserved patterns in protein chemistry may indeed exist across distant lineages. Such patterns in V3 loop amino acid chemistry may be indicative of stable lineages or convergence within this highly variable, though functionally and immunologically critical, region. We think that there may be parallels between the apparently stable HIV-2 V3 lineage and the previously mentioned HIV-1 V3 loops which are very similar at the protein level despite being distant by cladistic analysis, and which do not possess the distinctive positively charged residues. Highly conserved V3 loop protein sequences are also encountered in SIVAGMs and CIVs (chimpanzee viral strains), which do not appear to be pathogenic in their wild-caught natural hosts.

  15. Contrasting HIV phylogenetic relationships and V3 loop protein similarities

    SciTech Connect

    Korber, B. |; Myers, G.

    1992-12-31

    At least five distinct sequence subtypes of HIV-I can be identified from the major centers of the AMS pandemic. While it is too early to tell whether these subtypes are serologically or phenotypically similar or distinct in terms of properties such as pathogenicity and transmissibility, we can begin to investigate their potential for phenotypic divergence at the protein sequence level. Phylogenetic analysis of HIV DNA sequences is being widely used to examine lineages of different viral strains as they evolve and spread throughout the globe. We have identified five distinct HIV-1 subtypes (designated A-E), or clades, based on phylogenetic clustering patterns generated from genetic information from both the gag and envelope (env) genes from a spectrum of international isolates. Our initial observations concerning both HIV-1 and HIV-2 sequences indicate that conserved patterns in protein chemistry may indeed exist across distant lineages. Such patterns in V3 loop amino acid chemistry may be indicative of stable lineages or convergence within this highly variable, though functionally and immunologically critical, region. We think that there may be parallels between the apparently stable HIV-2 V3 lineage and the previously mentioned HIV-1 V3 loops which are very similar at the protein level despite being distant by cladistic analysis, and which do not possess the distinctive positively charged residues. Highly conserved V3 loop protein sequences are also encountered in SIVAGMs and CIVs (chimpanzee viral strains), which do not appear to be pathogenic in their wild-caught natural hosts.

  16. OSPREY: Protein Design with Ensembles, Flexibility, and Provable Algorithms

    PubMed Central

    Gainza, Pablo; Roberts, Kyle E.; Georgiev, Ivelin; Lilien, Ryan H.; Keedy, Daniel A.; Chen, Cheng-Yu; Reza, Faisal; Anderson, Amy C.; Richardson, David C.; Richardson, Jane S.; Donald, Bruce R.

    2013-01-01

    Summary We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) improved flexibility of the protein backbone, protein side chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally-optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe OSPREY, a protein redesign suite that implements our protein design algorithms. OSPREY has been used prospectively, with experimental validation, in several biomedically-relevant settings. We show in detail how OSPREY has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application. PMID:23422427

  17. Fast and anisotropic flexibility-rigidity index for protein flexibility and fluctuation analysis

    NASA Astrophysics Data System (ADS)

    Opron, Kristopher; Xia, Kelin; Wei, Guo-Wei

    2014-06-01

    Protein structural fluctuation, typically measured by Debye-Waller factors, or B-factors, is a manifestation of protein flexibility, which strongly correlates to protein function. The flexibility-rigidity index (FRI) is a newly proposed method for the construction of atomic rigidity functions required in the theory of continuum elasticity with atomic rigidity, which is a new multiscale formalism for describing excessively large biomolecular systems. The FRI method analyzes protein rigidity and flexibility and is capable of predicting protein B-factors without resorting to matrix diagonalization. A fundamental assumption used in the FRI is that protein structures are uniquely determined by various internal and external interactions, while the protein functions, such as stability and flexibility, are solely determined by the structure. As such, one can predict protein flexibility without resorting to the protein interaction Hamiltonian. Consequently, bypassing the matrix diagonalization, the original FRI has a computational complexity of O(N^2). This work introduces a fast FRI (fFRI) algorithm for the flexibility analysis of large macromolecules. The proposed fFRI further reduces the computational complexity to O(N). Additionally, we propose anisotropic FRI (aFRI) algorithms for the analysis of protein collective dynamics. The aFRI algorithms permit adaptive Hessian matrices, from a completely global 3N × 3N matrix to completely local 3 × 3 matrices. These 3 × 3 matrices, despite being calculated locally, also contain non-local correlation information. Eigenvectors obtained from the proposed aFRI algorithms are able to demonstrate collective motions. Moreover, we investigate the performance of FRI by employing four families of radial basis correlation functions. Both parameter optimized and parameter-free FRI methods are explored. Furthermore, we compare the accuracy and efficiency of FRI with some established approaches to flexibility analysis, namely, normal

  18. Fast and anisotropic flexibility-rigidity index for protein flexibility and fluctuation analysis

    SciTech Connect

    Opron, Kristopher; Xia, Kelin; Wei, Guo-Wei

    2014-06-21

    Protein structural fluctuation, typically measured by Debye-Waller factors, or B-factors, is a manifestation of protein flexibility, which strongly correlates to protein function. The flexibility-rigidity index (FRI) is a newly proposed method for the construction of atomic rigidity functions required in the theory of continuum elasticity with atomic rigidity, which is a new multiscale formalism for describing excessively large biomolecular systems. The FRI method analyzes protein rigidity and flexibility and is capable of predicting protein B-factors without resorting to matrix diagonalization. A fundamental assumption used in the FRI is that protein structures are uniquely determined by various internal and external interactions, while the protein functions, such as stability and flexibility, are solely determined by the structure. As such, one can predict protein flexibility without resorting to the protein interaction Hamiltonian. Consequently, bypassing the matrix diagonalization, the original FRI has a computational complexity of O(N{sup 2}). This work introduces a fast FRI (fFRI) algorithm for the flexibility analysis of large macromolecules. The proposed fFRI further reduces the computational complexity to O(N). Additionally, we propose anisotropic FRI (aFRI) algorithms for the analysis of protein collective dynamics. The aFRI algorithms permit adaptive Hessian matrices, from a completely global 3N × 3N matrix to completely local 3 × 3 matrices. These 3 × 3 matrices, despite being calculated locally, also contain non-local correlation information. Eigenvectors obtained from the proposed aFRI algorithms are able to demonstrate collective motions. Moreover, we investigate the performance of FRI by employing four families of radial basis correlation functions. Both parameter optimized and parameter-free FRI methods are explored. Furthermore, we compare the accuracy and efficiency of FRI with some established approaches to flexibility analysis, namely

  19. Effect of charge distribution in a flexible loop on the bioluminescence color of firefly luciferases.

    PubMed

    Moradi, Ali; Hosseinkhani, Saman; Naderi-Manesh, Hossein; Sadeghizadeh, Majid; Alipour, Bagher Said

    2009-01-27

    Firefly luciferase is a monooxygenase that catalyzes the ATP-dependent conversion of firefly luciferin into a luciferyl-adenylate, which is oxidized to an electronically excited oxyluciferin in a multistep reaction and produces visible light with a remarkable quantum yield. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetles, only luciferase from Phrixotrix railroad worm (Ph(RE)) emits red bioluminescence, naturally. The presence of Arg353 in Ph(RE) luciferase, which corresponds to the deleted residue in the other luciferases, is an important distinctive structural feature of it. Insertion of Arg356 into a green-emitter luciferase (Lampyris turkestanicus), corresponding to Arg353 in Phrixotrix hirtus, changed the emitted light from green to red. To further clarify the effect of this position on the light shift mechanism, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase, using site-specific insertion mutagenesis. Insertion of a residue with a positive side chain (Arg356 and Lys356) changed the light color to red, while insertion of a residue with a negative side chain (Glu356) had little effect on color. Insertion of a neutral residue (Gln356) at this position was performed without any change in bioluminescence spectra. Insertion of positively charged residues in this loop took place with a series of structural changes which were confirmed by fluorescence spectroscopy and homology modeling. Homology modeling reveals the appearance of a bulge in a flexible loop (T352-P359) upon mutation which shifts to the left side with a color change from green to red.

  20. The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters.

    PubMed

    Aseervatham, Jaya; Tran, Lucky; Machaca, Khaled; Boudker, Olga

    2015-01-01

    Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation.

  1. Understanding the challenges of protein flexibility in drug design.

    PubMed

    Antunes, Dinler A; Devaurs, Didier; Kavraki, Lydia E

    2015-12-01

    Protein-ligand interactions play key roles in various metabolic pathways, and the proteins involved in these interactions represent major targets for drug discovery. Molecular docking is widely used to predict the structure of protein-ligand complexes, and protein flexibility stands out as one of the most important and challenging issues for binding mode prediction. Various docking methods accounting for protein flexibility have been proposed, tackling problems of ever-increasing dimensionality. This paper presents an overview of conformational sampling methods treating target flexibility during molecular docking. Special attention is given to approaches considering full protein flexibility. Contrary to what is frequently done, this review does not rely on classical biomolecular recognition models to classify existing docking methods. Instead, it applies algorithmic considerations, focusing on the level of flexibility accounted for. This review also discusses the diversity of docking applications, from virtual screening (VS) of small drug-like compounds to geometry prediction (GP) of protein-peptide complexes. Considering the diversity of docking methods presented here, deciding which one is the best at treating protein flexibility depends on the system under study and the research application. In VS experiments, ensemble docking can be used to implicitly account for large-scale conformational changes, and selective docking can additionally consider local binding-site rearrangements. In other cases, on-the-fly exploration of the whole protein-ligand complex might be needed for accurate GP of the binding mode. Among other things, future methods are expected to provide alternative binding modes, which will better reflect the dynamic nature of protein-ligand interactions.

  2. The 1.59Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop.

    PubMed

    Raghunathan, Kannan; Vago, Frank S; Grindem, David; Ball, Terry; Wedemeyer, William J; Bagdasarian, Michael; Arvidson, Dennis N

    2014-02-01

    The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160-166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104-135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.

  3. Protein localization with flexible DNA or RNA.

    PubMed

    Bernhardsson, Sebastian; Mitarai, Namiko; Sneppen, Kim

    2012-01-01

    Localization of activity is ubiquitous in life, and also within sub-cellular compartments. Localization provides potential advantages as different proteins involved in the same cellular process may supplement each other on a fast timescale. It might also prevent proteins from being active in other regions of the cell. However localization is at odds with the spreading of unbound molecules by diffusion. We model the cost and gain for specific enzyme activity using localization strategies based on binding to sites of intermediate specificity. While such bindings in themselves decrease the activity of the protein on its target site, they may increase protein activity if stochastic motion allows the acting protein to touch both the intermediate binding site and the specific site simultaneously. We discuss this strategy in view of recent suggestions on long non-coding RNA as a facilitator of localized activity of chromatin modifiers.

  4. Functional Characterization of Antibodies against Neisseria gonorrhoeae Opacity Protein Loops

    PubMed Central

    Cole, Jessica G.; Jerse, Ann E.

    2009-01-01

    Background The development of a gonorrhea vaccine is challenged by the lack of correlates of protection. The antigenically variable neisserial opacity (Opa) proteins are expressed during infection and have a semivariable (SV) and highly conserved (4L) loop that could be targeted in a vaccine. Here we compared antibodies to linear (Ablinear) and cyclic (Abcyclic) peptides that correspond to the SV and 4L loops and selected hypervariable (HV2) loops for surface-binding and protective activity in vitro and in vivo. Methods/Findings AbSV cyclic bound a greater number of different Opa variants than AbSV linear, including variants that differed by seven amino acids. Antibodies to the 4L peptide did not bind Opa-expressing bacteria. AbSV cyclic and AbHV2 cyclic, but not AbSV linear or AbHV2 linear agglutinated homologous Opa variants, and AbHV2BD cyclic but not AbHV2BD linear blocked the association of OpaB variants with human endocervical cells. Only AbHV2BD linear were bactericidal against the serum resistant parent strain. Consistent with host restrictions in the complement cascade, the bactericidal activity of AbHV2BD linear was increased 8-fold when rabbit complement was used. None of the antibodies was protective when administered vaginally to mice. Antibody duration in the vagina was short-lived, however, with <50% of the antibodies recovered 3 hrs post-administration. Conclusions We conclude that an SV loop-specific cyclic peptide can be used to induce antibodies that recognize a broad spectrum of antigenically distinct Opa variants and have agglutination abilities. HV2 loop-specific cyclic peptides elicited antibodies with agglutination and adherence blocking abilities. The use of human complement when testing the bactericidal activity of vaccine-induced antibodies against serum resistant gonococci is also important. PMID:19956622

  5. On the modeling, and open loop control of a rotating thin flexible beam

    NASA Astrophysics Data System (ADS)

    Choura, Slim; Jayasuriya, Suhada; Medick, Matthew A.

    1989-12-01

    A set of governing differential equations is derived for the inplane motion of a rotating thin flexible beam. The beam is assumed to be linearly elastic and is connected to a rigid hub driven by a torque motor. Both flexural and extensional effects are included in the derivation. This coupling due to flexure and extension is usually neglected in studies dealing with the control of such a system. Models for typical control studies are often derived by utilizing an assumed mode approach where the mode shapes are obtained by solving the Euler-Bernoulli beam equation for flexural vibrations, with clamped-free or pinned-free boundary conditions. The coupled equations developed in this paper are used to demonstrate that typical models in control studies give satisfactory results up to a critical rotational speed. For the case where these coupled equations are specialized to simple flexure only, valid for low angular speeds, a unique feedforward control strategy can be derived. This is an open loop control strategy that enables total elimination of an a priori specified vibratory mode from the gross motion in a finite critical time.

  6. Applying Side-chain Flexibility in Motifs for Protein Docking

    PubMed Central

    Liu, Hui; Lin, Feng; Yang, Jian-Li; Wang, Hong-Rui; Liu, Xiu-Ling

    2015-01-01

    Conventional rigid docking algorithms have been unsatisfactory in their computational results, largely due to the fact that protein structures are flexible in live environments. In response, we propose to introduce the side-chain flexibility in protein motif into the docking. First, the Morse theory is applied to curvature labeling and surface region growing, for segmentation of the protein surface into smaller patches. Then, the protein is described by an ensemble of conformations that incorporate the flexibility of interface side chains and are sampled using rotamers. Next, a 3D rotation invariant shape descriptor is proposed to deal with the flexible motifs and surface patches; thus, pairwise complementarity matching is needed only between the convex patches of ligand and the concave patches of receptor. The iterative closest point (ICP) algorithm is implemented for geometric alignment of the two 3D protein surface patches. Compared with the fast Fourier transform-based global geometric matching algorithm and other methods, our FlexDock system generates much less false-positive docking results, which benefits identification of the complementary candidates. Our computational experiments show the advantages of the proposed flexible docking algorithm over its counterparts. PMID:26508871

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

    PubMed Central

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

    2015-01-01

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

  8. Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins.

    PubMed

    Blobel, Jascha; Brath, Ulrika; Bernadó, Pau; Diehl, Carl; Ballester, Lidia; Sornosa, Alejandra; Akke, Mikael; Pons, Miquel

    2011-12-01

    Addition of a 50 mM mixture of L: -arginine and L: -glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

  9. Calculating and scoring high quality multiple flexible protein structure alignments.

    PubMed

    Ritchie, David W

    2016-09-01

    Calculating multiple protein structure alignments (MSAs) is important for understanding functional and evolutionary relationships between protein families, and for modeling protein structures by homology. While incorporating backbone flexibility promises to circumvent many of the limitations of rigid MSA algorithms, very few flexible MSA algorithms exist today. This article describes several novel improvements to the Kpax algorithm which allow high quality flexible MSAs to be calculated. This article also introduces a new Gaussian-based MSA quality measure called 'M-score', which circumvents the pitfalls of RMSD-based quality measures. As well as calculating flexible MSAs, the new version of Kpax can also score MSAs from other aligners and from previously aligned reference datasets. Results are presented for a large-scale evaluation of the Homstrad, SABmark and SISY benchmark sets using Kpax and Matt as examples of state-of-the-art flexible aligners and 3DCOMB as an example of a state-of-the-art rigid aligner. These results demonstrate the utility of the M-score as a measure of MSA quality and show that high quality MSAs may be achieved when structural flexibility is properly taken into account. Kpax 5.0 may be downloaded for academic use at http://kpax.loria.fr/ dave.ritchie@inria.fr Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. The flexibility and dynamics of protein disulfide isomerase

    PubMed Central

    Wells, Stephen A.; Emilio Jimenez‐Roldan, J.; Bhattacharyya, Moitrayee; Vishweshwara, Saraswathi; Freedman, Robert B.

    2016-01-01

    ABSTRACT We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse‐graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures—and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776–1785. © 2016 Wiley Periodicals, Inc. PMID:27616289

  11. Protein conformational flexibility prediction using machine learning

    PubMed Central

    Trott, Oleg; Siggers, Keri; Rost, Burkhard; Palmer, Arthur G.

    2008-01-01

    Using a data set of 16 proteins, a neural network has been trained to predict backbone 15N generalized order parameters from the three-dimensional structures of proteins. The final network parameterization contains six input features. The average prediction accuracy, as measured by the Pearson correlation coefficient between experimental and predicted values of the square of the generalized order parameter is > 0.70. Predicted order parameters for non-terminal amino acid residues depends most strongly on local packing density and the probability that the residue is located in regular secondary structure. PMID:18313957

  12. Rigidity versus flexibility: the dilemma of understanding protein thermal stability.

    PubMed

    Karshikoff, Andrey; Nilsson, Lennart; Ladenstein, Rudolf

    2015-10-01

    The role of fluctuations in protein thermostability has recently received considerable attention. In the current literature a dualistic picture can be found: thermostability seems to be associated with enhanced rigidity of the protein scaffold in parallel with the reduction of flexible parts of the structure. In contradiction to such arguments it has been shown by experimental studies and computer simulation that thermal tolerance of a protein is not necessarily correlated with the suppression of internal fluctuations and mobility. Both concepts, rigidity and flexibility, are derived from mechanical engineering and represent temporally insensitive features describing static properties, neglecting that relative motion at certain time scales is possible in structurally stable regions of a protein. This suggests that a strict separation of rigid and flexible parts of a protein molecule does not describe the reality correctly. In this work the concepts of mobility/flexibility versus rigidity will be critically reconsidered by taking into account molecular dynamics calculations of heat capacity and conformational entropy, salt bridge networks, electrostatic interactions in folded and unfolded states, and the emerging picture of protein thermostability in view of recently developed network theories. Last, but not least, the influence of high temperature on the active site and activity of enzymes will be considered. © 2015 FEBS.

  13. A Binding Model and Similarity for Flexible Modular Proteins

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Feinauer, Christoph J.; Hofmann, Andreas; Goldt, Sebastian; Liu, Lei; Heermann, Dieter W.

    2013-03-01

    Modular proteins are one of the most commonly found disordered protein motifs. An example is CTCF, a protein that has been named the master waver of the genome i.e., the organizer of the 3D structure of the chromosomes. Using NMR and numerical simulations, much progress has been made in understanding their various functions and ways of binding. Modular proteins are often composed of protein modules interconnected by flexible linkers. They can be imagined as ``beads on a string.'' We argue that when the number of beads is small, these structures behave like a self avoiding random walk. Nevertheless, when binding to a target, linkers can fold in more ordered and stable states. At the same time, folding can influence functional roles. We show that the flexibility of the linkers can boost binding affinity. As a result of flexibility, the conformations of these proteins before and after binding are different. So this implies that generic binding site prediction methods may fail. To deal with this we introduce a new methodology to characterize and compare these flexible structures. Employing topological concepts we propose a method which intrinsically fuses topology and geometry. GM gratefully acknowledges support from the HGS-MathComp and the RTG 1653.

  14. Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions.

    PubMed

    Najibi, Seyed Morteza; Maadooliat, Mehdi; Zhou, Lan; Huang, Jianhua Z; Gao, Xin

    2017-01-01

    Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

  15. External loops at the ferredoxin-NADP(+) reductase protein-partner binding cavity contribute to substrates allocation.

    PubMed

    Sánchez-Azqueta, Ana; Martínez-Júlvez, Marta; Hervás, Manuel; Navarro, José A; Medina, Milagros

    2014-02-01

    Ferredoxin-NADP(+) reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)(+)/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet-loop-sheet motif, loop102-114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261-269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102-114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261-269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261-269 appears a determinant to ensure reversibility in photosynthetic FNRs. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Switch-Loop Flexibility Affects Transport of Large Drugs by the Promiscuous AcrB Multidrug Efflux Transporter

    PubMed Central

    Cha, Hi-jea; Müller, Reinke T.

    2014-01-01

    Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å2 are less well transported than other substrates. PMID:24914123

  17. How does symmetry impact the flexibility of proteins?

    PubMed Central

    Schulze, Bernd; Sljoka, Adnan; Whiteley, Walter

    2014-01-01

    It is well known that (i) the flexibility and rigidity of proteins are central to their function, (ii) a number of oligomers with several copies of individual protein chains assemble with symmetry in the native state and (iii) added symmetry sometimes leads to added flexibility in structures. We observe that the most common symmetry classes of protein oligomers are also the symmetry classes that lead to increased flexibility in certain three-dimensional structures—and investigate the possible significance of this coincidence. This builds on the well-developed theory of generic rigidity of body–bar frameworks, which permits an analysis of the rigidity and flexibility of molecular structures such as proteins via fast combinatorial algorithms. In particular, we outline some very simple counting rules and possible algorithmic extensions that allow us to predict continuous symmetry-preserving motions in body–bar frameworks that possess non-trivial point-group symmetry. For simplicity, we focus on dimers, which typically assemble with twofold rotational axes, and often have allosteric function that requires motions to link distant sites on the two protein chains. PMID:24379431

  18. Degradation of microinjected proteins: the role of substrate flexibility

    SciTech Connect

    Rote, K.V.

    1985-01-01

    RB-mediated microinjection was used to introduce radioiodinated proteins of similar structure, but diverse flexibilities, into HeLa cells. Rates of intracellular degradation were then measured by release of /sup 125/I-tyrosine into the media. Ribonuclease-A was much more stable to degradation by trypsin, pepsin, or papain than its relatively flexible derivatives ribonuclease-S and S-protein. Likewise, ribonuclease-S and S-protein were degraded more quickly in reticulocyte lysates than ribonuclease-A. In contrast, all three proteins displayed similar, if not identical, half-lives in vivo. Similarly, intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were in the same range whether they were measured in the free state or following complex formation, which drastically decreases flexibility. Trypsinogen, which contains a relatively flexible activation domain, was degraded more slowly than anhydrotrypsin. Nondenaturing agarose or polyacrylamide gel electrophoresis of microinjected cell lysates revealed that complexes of trypsin and its inhibitors remained intact following radioiodination and introduction into cells, and are therefore degraded as a unit. All microinjected proteins remained in their unbound, unprocessed forms prior to degradation.

  19. How does symmetry impact the flexibility of proteins?

    PubMed

    Schulze, Bernd; Sljoka, Adnan; Whiteley, Walter

    2014-02-13

    It is well known that (i) the flexibility and rigidity of proteins are central to their function, (ii) a number of oligomers with several copies of individual protein chains assemble with symmetry in the native state and (iii) added symmetry sometimes leads to added flexibility in structures. We observe that the most common symmetry classes of protein oligomers are also the symmetry classes that lead to increased flexibility in certain three-dimensional structures-and investigate the possible significance of this coincidence. This builds on the well-developed theory of generic rigidity of body-bar frameworks, which permits an analysis of the rigidity and flexibility of molecular structures such as proteins via fast combinatorial algorithms. In particular, we outline some very simple counting rules and possible algorithmic extensions that allow us to predict continuous symmetry-preserving motions in body-bar frameworks that possess non-trivial point-group symmetry. For simplicity, we focus on dimers, which typically assemble with twofold rotational axes, and often have allosteric function that requires motions to link distant sites on the two protein chains.

  20. A flexible fiber displacement sensor with tunable resolution and dynamic range based on a few-mode fiber loop

    NASA Astrophysics Data System (ADS)

    Fu, Xin; Lu, Ping; Liu, Deming; Zhang, Jiangshan; Jiang, Shibin

    2017-02-01

    In this article, we propose a fiber displacement sensor based on a few mode fiber loop sandwiched between two single mode fibers (SMF). The proposed sensor is flexible due to the tunable resolution and dynamic range. The FMF is coiled to a fiber loop by making a knot. The in-line MZI sensing structure is fixed on a two dimensional (2D) translation stages. By moving one stage while another stage is fixed, the displacement is applied on the sensing structure. The resolution of the translation stage is 10μm. The few mode fiber loop acts as the transducer for the displacement sensing. The displacement will change the radius of the few mode fiber loop, which leads to a wavelength shift of the interference pattern. When the fiber loop has different initial radius, the same displacement will cause a different curvature variation. So the sensitivity of the wavelength shift to the displacement is dependent on the initial radius. A smaller initial radius of the loop will lead to a larger sensitivity, higher resolution but smaller dynamic range, so it is proper for micro displacement sensing. On the contrary is the lager initial radius that is proper for sensing in a large dynamic range. By simply adjusting the initial radius of the transducer loop, different sensitivity and resolution can be reached. Experimental results show the sensitivities of 0.267nm/mm, 0.384nm/mm, 0.749nm/mm and 1.06nm/mm for initial loop radius of 1.9cm, 1.5cm, 1cm and 0.75cm, respectively.

  1. Conformational flexibility of loops of myosin enhances the global bias in the actin-myosin interaction landscape.

    PubMed

    Nie, Qing-Miao; Sasai, Masaki; Terada, Tomoki P

    2014-04-14

    A long-standing controversy on the mechanism of an actomyosin motor is the role of the Brownian motion of the myosin head in force generation. In order to shed light on this problem, we calculate free-energy landscapes of interaction between an actin filament and the head (S1) of myosin II by using a coarse-grained model of actomyosin. The results show that the free-energy landscape has a global gradient toward the strong-binding site on actin filament, which explains the biased Brownian motion of myosin S1 observed in a single-molecule experiment [Kitamura et al., Nature, 1999, 397, 129 and Biophysics, 2005, 1, 1]. The distinct global gradient in the landscape is brought about only when the conformation of loop 2 at the actin interface of myosin S1 is flexible. The conformational flexibility of loop 3 also contributes to the gradient in the landscape by compensating the role of loop 2. Though the structure of loop 2 is expanded in the weak-binding state, loop 2 shows the larger fluctuation of compaction and expansion due to the actin-myosin interactions as myosin S1 moves toward the strong-binding site on actin filament. Hence, the increase in the compaction-expansion fluctuation of loop 2, the stronger binding of myosin to actin, and the biased Brownian motion of myosin S1 are coupled with each other and should take place in a concurrent way. This predicted coupling should provide opportunities to further test the hypothesis of the biased Brownian motion in actomyosin.

  2. Flexible, symmetry-directed approach to assembling protein cages

    PubMed Central

    Sciore, Aaron; Su, Min; Koldewey, Philipp; Eschweiler, Joseph D.; Diffley, Kelsey A.; Linhares, Brian M.; Ruotolo, Brandon T.; Bardwell, James C. A.; Skiniotis, Georgios; Marsh, E. Neil G.

    2016-01-01

    The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries. PMID:27432965

  3. Flexible, symmetry-directed approach to assembling protein cages.

    PubMed

    Sciore, Aaron; Su, Min; Koldewey, Philipp; Eschweiler, Joseph D; Diffley, Kelsey A; Linhares, Brian M; Ruotolo, Brandon T; Bardwell, James C A; Skiniotis, Georgios; Marsh, E Neil G

    2016-08-02

    The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.

  4. Fast and anisotropic flexibility-rigidity index for protein flexibility and fluctuation analysis

    PubMed Central

    Opron, Kristopher; Xia, Kelin; Wei, Guo-Wei

    2014-01-01

    Protein structural fluctuation, typically measured by Debye-Waller factors, or B-factors, is a manifestation of protein flexibility, which strongly correlates to protein function. The flexibility-rigidity index (FRI) is a newly proposed method for the construction of atomic rigidity functions required in the theory of continuum elasticity with atomic rigidity, which is a new multiscale formalism for describing excessively large biomolecular systems. The FRI method analyzes protein rigidity and flexibility and is capable of predicting protein B-factors without resorting to matrix diagonalization. A fundamental assumption used in the FRI is that protein structures are uniquely determined by various internal and external interactions, while the protein functions, such as stability and flexibility, are solely determined by the structure. As such, one can predict protein flexibility without resorting to the protein interaction Hamiltonian. Consequently, bypassing the matrix diagonalization, the original FRI has a computational complexity of \\documentclass[12pt]{minimal}\\begin{document}${\\cal O}(N^2)$\\end{document}O(N2). This work introduces a fast FRI (fFRI) algorithm for the flexibility analysis of large macromolecules. The proposed fFRI further reduces the computational complexity to \\documentclass[12pt]{minimal}\\begin{document}${\\cal O}(N)$\\end{document}O(N). Additionally, we propose anisotropic FRI (aFRI) algorithms for the analysis of protein collective dynamics. The aFRI algorithms permit adaptive Hessian matrices, from a completely global 3N × 3N matrix to completely local 3 × 3 matrices. These 3 × 3 matrices, despite being calculated locally, also contain non-local correlation information. Eigenvectors obtained from the proposed aFRI algorithms are able to demonstrate collective motions. Moreover, we investigate the performance of FRI by employing four families of radial basis correlation functions. Both parameter optimized and parameter-free FRI

  5. Interplay between Protein Thermal Flexibility and Kinetic Stability.

    PubMed

    Quezada, Andrea G; Díaz-Salazar, A Jessica; Cabrera, Nallely; Pérez-Montfort, Ruy; Piñeiro, Ángel; Costas, Miguel

    2017-01-03

    Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (β/α)8 barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.

  6. Prion protein β2-α2 loop conformational landscape.

    PubMed

    Caldarulo, Enrico; Barducci, Alessandro; Wüthrich, Kurt; Parrinello, Michele

    2017-09-05

    In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal "cellular" prion protein ([Formula: see text]) is transformed into amyloid aggregates representing the "scrapie form" of the protein ([Formula: see text]). Continued research on this system is of keen interest, since new information on the physiological function of [Formula: see text] in healthy organisms is emerging, as well as new data on the mechanism of the transformation of [Formula: see text] to [Formula: see text] In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of [Formula: see text] The focus of the data analysis was on an 11-residue polypeptide segment in mouse [Formula: see text](121-231) that includes the [Formula: see text]2-[Formula: see text]2 loop of residues 167-170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse [Formula: see text] and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on [Formula: see text], providing quantitative insights into the nature of the structural transition-related function of the [Formula: see text]2-[Formula: see text]2 loop.

  7. Flexible and rigid structures in HIV-1 p17 matrix protein monitored by relaxation and amide proton exchange with NMR.

    PubMed

    Ohori, Yuka; Okazaki, Honoka; Watanabe, Satoru; Tochio, Naoya; Arai, Munehito; Kigawa, Takanori; Nishimura, Chiaki

    2014-03-01

    The HIV-1 p17 matrix protein is a multifunctional protein that interacts with other molecules including proteins and membranes. The dynamic structure between its folded and partially unfolded states can be critical for the recognition of interacting molecules. One of the most important roles of the p17 matrix protein is its localization to the plasma membrane with the Gag polyprotein. The myristyl group attached to the N-terminus on the p17 matrix protein functions as an anchor for binding to the plasma membrane. Biochemical studies revealed that two regions are important for its function: D14-L31 and V84-V88. Here, the dynamic structures of the p17 matrix protein were studied using NMR for relaxation and amide proton exchange experiments at the physiological pH of 7.0. The results revealed that the α12-loop, which includes the 14-31 region, was relatively flexible, and that helix 4, including the 84-88 region, was the most protected helix in this protein. However, the residues in the α34-loop near helix 4 had a low order parameter and high exchange rate of amide protons, indicating high flexibility. This region is probably flexible because this loop functions as a hinge for optimizing the interactions between helices 3 and 4. The C-terminal long region of K113-Y132 adopted a disordered structure. Furthermore, the C-terminal helix 5 appeared to be slightly destabilized due to the flexible C-terminal tail based on the order parameters. Thus, the dynamic structure of the p17 matrix protein may be related to its multiple functions.

  8. Joining RDC data from flexible protein domains

    NASA Astrophysics Data System (ADS)

    Sgheri, Luca

    2010-11-01

    We study the inverse problem of determining the conformational freedom of two protein domains from residual dipolar coupling (RDC) measurements. For each paramagnetic ion attached to one of the domains we obtain a magnetic susceptibility tensor χ from the RDC of couples of atoms of that domain, and a mean paramagnetic susceptibility tensor {\\bar{\\chi }} from the RDC of couples of atoms of the other domain. The latter is an integral average of rotations of χ which depends on the conformational freedom of the two domains. In this paper we consider the case when we have data from paramagnetic ions attached separately to each of the domains. We prove that in this case not all the elements of χ and {\\bar{\\chi }} are independent. We derive the mathematical equations for the compatibility of the measurements and show how these relations can be used in the presence of noisy data to determine a compatible set of χ and {\\bar{\\chi }} with an unconstrained minimization. If available, information about the shape of the noise can be included in the target function. We show that in this case the compatible set obtained has a reduced error with respect to the noisy data.

  9. Alteration of the flexible loop in 1-deoxy-D-xylulose-5-phosphate reductoisomerase boosts enthalpy-driven inhibition by fosmidomycin.

    PubMed

    Kholodar, Svetlana A; Tombline, Gregory; Liu, Juan; Tan, Zhesen; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

    2014-06-03

    1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy-enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin's phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.

  10. OSPREY: protein design with ensembles, flexibility, and provable algorithms.

    PubMed

    Gainza, Pablo; Roberts, Kyle E; Georgiev, Ivelin; Lilien, Ryan H; Keedy, Daniel A; Chen, Cheng-Yu; Reza, Faisal; Anderson, Amy C; Richardson, David C; Richardson, Jane S; Donald, Bruce R

    2013-01-01

    We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) improved flexibility of the protein backbone, protein side-chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe OSPREY, a protein redesign suite that implements our protein design algorithms. OSPREY has been used prospectively, with experimental validation, in several biomedically relevant settings. We show in detail how OSPREY has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application. OSPREY is free and open source under a Lesser GPL license. The latest version is OSPREY 2.0. The program, user manual, and source code are available at www.cs.duke.edu/donaldlab/software.php. osprey@cs.duke.edu. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping

    SciTech Connect

    Giovan, Stefan M.; Scharein, Robert G.; Hanke, Andreas; Levene, Stephen D.

    2014-11-07

    We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

  12. Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping

    NASA Astrophysics Data System (ADS)

    Giovan, Stefan M.; Scharein, Robert G.; Hanke, Andreas; Levene, Stephen D.

    2014-11-01

    We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

  13. Investigation of Catalytic Loop Structure, Dynamics and Function Relationship of Yersinia Protein Tyrosine Phosphatase by Temperature-Jump Relaxation Spectroscopy and X-ray Structural Determination

    PubMed Central

    Ke, Shan; Ho, Meng-Chiao; Zhadin, Nickolay; Deng, Hua; Callender, Robert

    2012-01-01

    Yersinia Protein Tyrosine Phosphatase (YopH) is the most efficient enzyme amongst all PTPases and YopH is hyperactive compared to human PTPases, interferes with mammalian cellular pathways to achieve the pathogenicity of Yersinia. Two properties related to the catalytic loop structure differences have been proposed to affect its dynamics and enzyme efficiency. One is the ability of the loop to form stabilizing interactions to bound ligand after loop closure, which has long been recognized. In addition, the loop flexibility/mobility was suggested in a previous study to be a factor as well, based on the observation that incremental changes in PTPase loop structure by single point mutations to alanine often induce incremental changes in enzyme catalytic efficiency. In this study, the temperature jump relaxation spectroscopy (T-jump) has been used to discern the subtle changes of the loop dynamics due to point loop mutations. As expected, our results suggest a correlation between loop dynamics and the size of the residue on the catalytic loop. The stabilization of the enzyme-ligand complex is often enthalpy driven, achieved by formation of additional favorable hydrogen bonding/ionic interactions after loop closure. Interestingly, our T-jump and X-ray crystallography studies on YopH suggest that the elimination of some ligand-protein interactions by mutation does not necessarily destabilize the ligand-enzyme complex after loop closure since the increased entropy in the forms of more mobile protein residues may be sufficient to compensate the free energy loss due to lost interactions and may even lead to enhanced efficiency of the enzyme catalysis. How these competing loop properties may affect loop dynamics and enzyme function are discussed. PMID:22564106

  14. Peierls-Nabarro barrier and protein loop propagation.

    PubMed

    Sieradzan, Adam K; Niemi, Antti; Peng, Xubiao

    2014-12-01

    When a self-localized quasiparticle excitation propagates along a discrete one-dimensional lattice, it becomes subject to a dissipation that converts the kinetic energy into lattice vibrations. Eventually the kinetic energy no longer enables the excitation to cross over the minimum energy barrier between neighboring sites, and the excitation becomes localized within a lattice cell. In the case of a protein, the lattice structure consists of the C(α) backbone. The self-localized quasiparticle excitation is the elemental building block of loops. It can be modeled by a kink that solves a variant of the discrete nonlinear Schrödinger equation. We study the propagation of such a kink in the case of the protein G related albumin-binding domain, using the united residue coarse-grained molecular-dynamics force field. We estimate the height of the energy barriers that the kink needs to cross over in order to propagate along the backbone lattice. We analyze how these barriers give rise to both stresses and reliefs, which control the kink movement. For this, we deform a natively folded protein structure by parallel translating the kink along the backbone away from its native position. We release the transposed kink, and we follow how it propagates along the backbone toward the native location. We observe that the dissipative forces that are exerted on the kink by the various energy barriers have a pivotal role in determining how a protein folds toward its native state.

  15. Flexibility.

    ERIC Educational Resources Information Center

    Humphrey, L. Dennis

    1981-01-01

    Flexibility is an important aspect of all sports and recreational activities. Flexibility can be developed and maintained by stretching exercises. Exercises designed to develop flexibility in ankle joints, knees, hips, and the lower back are presented. (JN)

  16. Flexibility.

    ERIC Educational Resources Information Center

    Humphrey, L. Dennis

    1981-01-01

    Flexibility is an important aspect of all sports and recreational activities. Flexibility can be developed and maintained by stretching exercises. Exercises designed to develop flexibility in ankle joints, knees, hips, and the lower back are presented. (JN)

  17. Size, shape, and flexibility of proteins and DNA.

    PubMed

    Rawat, Nidhi; Biswas, Parbati

    2009-10-28

    Size, shape, and flexibility are the important topological parameters which characterize the functional specificity and different types of interactions in proteins and DNA. The size of proteins and DNA, often measured by the radius of gyration (R(G)), are determined from the coordinates of their respective structures available in Protein Data Bank and Nucleic Acid Data Bank. The mean square radius of gyration obeys Flory's scaling law given by R(G) (2) approximately N(2nu) where N is the number of amino acid residues/nucleotides. The scaling exponent nu reflects the different characteristic features of nonglobular proteins, natively unstructured proteins, and DNA. The asymmetry in the shapes of proteins and DNA are investigated using the asphericity (Delta) parameter and the shape parameter (S), calculated from the eigenvalues of the moment of inertia tensor. The distributions of Delta and S show that most nonglobular proteins and DNA are aspherical and prolate (S>0). Natively unstructured proteins are comparatively spherically symmetrical having both prolate and oblate shapes. The flexibility of these molecules is characterized by the persistence length (l(p)). Persistence length for natively unstructured proteins is determined by fitting the distance distribution function P(r) to the wormlike chain (WLC) model in the limit of r>R(G). For nonglobular proteins and DNA, l(p) may be computed from the Benoit-Doty approximation for unperturbed radius of gyration of the WLC. The flexibilities of the proteins and DNA increases with the chain length. This is due to an increase in the nonlocal interactions with the increase in N, needed to minimize the conformational fluctuations in the native state. The persistence length of these proteins has not yet been measured directly. Analysis of the two-body contacts for the proteins reveals that the nonglobular proteins are less densely packed compared to the natively unstructured proteins with least side-chain side chain

  18. Docking validation resources: protein family and ligand flexibility experiments.

    PubMed

    Mukherjee, Sudipto; Balius, Trent E; Rizzo, Robert C

    2010-11-22

    A database consisting of 780 ligand-receptor complexes, termed SB2010, has been derived from the Protein Databank to evaluate the accuracy of docking protocols for regenerating bound ligand conformations. The goal is to provide easily accessible community resources for development of improved procedures to aid virtual screening for ligands with a wide range of flexibilities. Three core experiments using the program DOCK, which employ rigid (RGD), fixed anchor (FAD), and flexible (FLX) protocols, were used to gauge performance by several different metrics: (1) global results, (2) ligand flexibility, (3) protein family, and (4) cross-docking. Global spectrum plots of successes and failures vs rmsd reveal well-defined inflection regions, which suggest the commonly used 2 Å criteria is a reasonable choice for defining success. Across all 780 systems, success tracks with the relative difficulty of the calculations: RGD (82.3%) > FAD (78.1%) > FLX (63.8%). In general, failures due to scoring strongly outweigh those due to sampling. Subsets of SB2010 grouped by ligand flexibility (7-or-less, 8-to-15, and 15-plus rotatable bonds) reveal that success degrades linearly for FAD and FLX protocols, in contrast to RGD, which remains constant. Despite the challenges associated with FLX anchor orientation and on-the-fly flexible growth, success rates for the 7-or-less (74.5%) and, in particular, the 8-to-15 (55.2%) subset are encouraging. Poorer results for the very flexible 15-plus set (39.3%) indicate substantial room for improvement. Family-based success appears largely independent of ligand flexibility, suggesting a strong dependence on the binding site environment. For example, zinc-containing proteins are generally problematic, despite moderately flexible ligands. Finally, representative cross-docking examples, for carbonic anhydrase, thermolysin, and neuraminidase families, show the utility of family-based analysis for rapid identification of particularly good or bad

  19. DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics

    PubMed Central

    Laurens, Niels; Rusling, David A.; Pernstich, Christian; Brouwer, Ineke; Halford, Stephen E.; Wuite, Gijs J. L.

    2012-01-01

    Protein-induced DNA looping is crucial for many genetic processes such as transcription, gene regulation and DNA replication. Here, we use tethered-particle motion to examine the impact of DNA bending and twisting rigidity on loop capture and release, using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric sequence, invariably aligning the sites in parallel. On account of the fixed alignment, the topology of the DNA loop is set by the orientation of the sites along the DNA. We show that both the separation of the FokI sites and their orientation, altering, respectively, the twisting and the bending of the DNA needed to juxtapose the sites, have profound effects on the dynamics of the looping interaction. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, the introduction of a 4-nt gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to stabilise its DNA-looping interaction by acting as a ‘torsional’ catch bond. PMID:22373924

  20. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

    SciTech Connect

    Meshcheryakov, Vladimir A.; Kitao, Akio; Matsunami, Hideyuki; Samatey, Fadel A.

    2013-05-01

    Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, the crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.

  1. Highly Flexible Protein-Peptide Docking Using CABS-Dock.

    PubMed

    Ciemny, Maciej Paweł; Kurcinski, Mateusz; Kozak, Konrad Jakub; Kolinski, Andrzej; Kmiecik, Sebastian

    2017-01-01

    Protein-peptide molecular docking is a difficult modeling problem. It is even more challenging when significant conformational changes that may occur during the binding process need to be predicted. In this chapter, we demonstrate the capabilities and features of the CABS-dock server for flexible protein-peptide docking. CABS-dock allows highly efficient modeling of full peptide flexibility and significant flexibility of a protein receptor. During CABS-dock docking, the peptide folding and binding process is explicitly simulated and no information about the peptide binding site or its structure is used. This chapter presents a successful CABS-dock use for docking a potentially therapeutic peptide to a protein target. Moreover, simulation contact maps, a new CABS-dock feature, are described and applied to the docking test case. Finally, a tutorial for running CABS-dock from the command line or command line scripts is provided. The CABS-dock web server is available from http://biocomp.chem.uw.edu.pl/CABSdock/ .

  2. A local average distance descriptor for flexible protein structure comparison

    PubMed Central

    2014-01-01

    Background Protein structures are flexible and often show conformational changes upon binding to other molecules to exert biological functions. As protein structures correlate with characteristic functions, structure comparison allows classification and prediction of proteins of undefined functions. However, most comparison methods treat proteins as rigid bodies and cannot retrieve similarities of proteins with large conformational changes effectively. Results In this paper, we propose a novel descriptor, local average distance (LAD), based on either the geodesic distances (GDs) or Euclidean distances (EDs) for pairwise flexible protein structure comparison. The proposed method was compared with 7 structural alignment methods and 7 shape descriptors on two datasets comprising hinge bending motions from the MolMovDB, and the results have shown that our method outperformed all other methods regarding retrieving similar structures in terms of precision-recall curve, retrieval success rate, R-precision, mean average precision and F1-measure. Conclusions Both ED- and GD-based LAD descriptors are effective to search deformed structures and overcome the problems of self-connection caused by a large bending motion. We have also demonstrated that the ED-based LAD is more robust than the GD-based descriptor. The proposed algorithm provides an alternative approach for blasting structure database, discovering previously unknown conformational relationships, and reorganizing protein structure classification. PMID:24694083

  3. E-proteins and ID-proteins: Helix-loop-helix partners in development and disease

    PubMed Central

    Wang, Lan-Hsin; Baker, Nicholas E.

    2015-01-01

    The basic Helix-Loop-Helix (bHLH) proteins represent a well-known class of transcriptional regulators. Many bHLH proteins act as heterodimers with members of a class of ubiquitous partners, the E-proteins. A widely-expressed class of inhibitory heterodimer partners- the Inhibitor of DNA-binding (ID) proteins- also exists. Genetic and molecular analyses in humans and in knockout mice implicate E-proteins and ID-proteins in a wide variety of diseases, belying the notion that they are non-specific partner proteins. Here, we explore relationships of E-proteins and ID-proteins to a variety of disease processes and highlight gaps in knowledge of disease mechanisms. PMID:26555048

  4. Persistent homology analysis of protein structure, flexibility and folding

    PubMed Central

    Xia, Kelin; Wei, Guo-Wei

    2014-01-01

    Proteins are the most important biomolecules for living organisms. The understanding of protein structure, function, dynamics and transport is one of most challenging tasks in biological science. In the present work, persistent homology is, for the first time, introduced for extracting molecular topological fingerprints (MTFs) based on the persistence of molecular topological invariants. MTFs are utilized for protein characterization, identification and classification. The method of slicing is proposed to track the geometric origin of protein topological invariants. Both all-atom and coarse-grained representations of MTFs are constructed. A new cutoff-like filtration is proposed to shed light on the optimal cutoff distance in elastic network models. Based on the correlation between protein compactness, rigidity and connectivity, we propose an accumulated bar length generated from persistent topological invariants for the quantitative modeling of protein flexibility. To this end, a correlation matrix based filtration is developed. This approach gives rise to an accurate prediction of the optimal characteristic distance used in protein B-factor analysis. Finally, MTFs are employed to characterize protein topological evolution during protein folding and quantitatively predict the protein folding stability. An excellent consistence between our persistent homology prediction and molecular dynamics simulation is found. This work reveals the topology-function relationship of proteins. PMID:24902720

  5. Glycerol effects on protein flexibility: a tryptophan phosphorescence study.

    PubMed Central

    Gonnelli, M.; Strambini, G. B.

    1993-01-01

    In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule. PMID:8369422

  6. Identifying the adaptive mechanism in globular proteins: Fluctuations in densely packed regions manipulate flexible parts

    NASA Astrophysics Data System (ADS)

    Yilmaz, Lutfu Safak; Atilgan, Ali Rana

    2000-09-01

    A low-resolution structural model based on the packing geometry of α-carbons is utilized to establish a connection between the flexible and rigid parts of a folded protein. The former commonly recognizes a complementing molecule for making a complex, while the latter manipulates the necessary conformational change for binding. We attempt analytically to distinguish this control architecture that intrinsically exists in globular proteins. First with two-dimensional simple models, then for a native protein, bovine pancreatic trypsin inhibitor, we explicitly demonstrate that inserting fluctuations in tertiary contacts supported by the stable core, one can regulate the displacement of residues on loop regions. The positional fluctuations of the flexible regions are annihilated by the rest of the protein in conformity with the Le Chatelier-Braun principle. The results indicate that the distortion of the principal nonbonded contacts between highly packed residues is accompanied by that of the slavery fluctuations that are widely distributed over the native structure. These positional arrangements do not appear in a reciprocal relation between a perturbation and the associated response; the effect of a movement of residue i on residue j is not equal to that of the same movement of residue j on residue i.

  7. SODOCK: swarm optimization for highly flexible protein-ligand docking.

    PubMed

    Chen, Hung-Ming; Liu, Bo-Fu; Huang, Hui-Ling; Hwang, Shiow-Fen; Ho, Shinn-Ying

    2007-01-30

    Protein-ligand docking can be formulated as a parameter optimization problem associated with an accurate scoring function, which aims to identify the translation, orientation, and conformation of a docked ligand with the lowest energy. The parameter optimization problem for highly flexible ligands with many rotatable bonds is more difficult than that for less flexible ligands using genetic algorithm (GA)-based approaches, due to the large numbers of parameters and high correlations among these parameters. This investigation presents a novel optimization algorithm SODOCK based on particle swarm optimization (PSO) for solving flexible protein-ligand docking problems. To improve efficiency and robustness of PSO, an efficient local search strategy is incorporated into SODOCK. The implementation of SODOCK adopts the environment and energy function of AutoDock 3.05. Computer simulation results reveal that SODOCK is superior to the Lamarckian genetic algorithm (LGA) of AutoDock, in terms of convergence performance, robustness, and obtained energy, especially for highly flexible ligands. The results also reveal that PSO is more suitable than the conventional GA in dealing with flexible docking problems with high correlations among parameters. This investigation also compared SODOCK with four state-of-the-art docking methods, namely GOLD 1.2, DOCK 4.0, FlexX 1.8, and LGA of AutoDock 3.05. SODOCK obtained the smallest RMSD in 19 of 37 cases. The average 2.29 A of the 37 RMSD values of SODOCK was better than those of other docking programs, which were all above 3.0 A.

  8. Protein-mediated loops and phase transition in nonthermal denaturation of DNA

    NASA Astrophysics Data System (ADS)

    Petrosyan, Karen G.; Hu, Chin-Kun

    2011-01-01

    We use a statistical mechanical model to study nonthermal denaturation of DNA in the presence of protein-mediated loops. We find that looping proteins which randomly link DNA bases located at a distance along the chain could cause a first-order phase transition. We estimate the denaturation transition time near the phase transition, which can be compared with experimental data. The model describes the formation of multiple loops via dynamical (fluctuational) linking between looping proteins, which is essential in many cellular biological processes.

  9. A chemical chaperone induces inhomogeneous conformational changes in flexible proteins.

    PubMed

    Hamdane, Djemel; Velours, Christophe; Cornu, David; Nicaise, Magali; Lombard, Murielle; Fontecave, Marc

    2016-07-27

    Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the

  10. Protein-based flexible whispering gallery mode resonators

    NASA Astrophysics Data System (ADS)

    Yilmaz, Huzeyfe; Pena-Francesch, Abdon; Xu, Linhua; Shreiner, Robert; Jung, Huihun; Huang, Steven H.; Özdemir, Sahin K.; Demirel, Melik C.; Yang, Lan

    2016-02-01

    The idea of creating photonics tools for sensing, imaging and material characterization has long been pursued and many achievements have been made. Approaching the level of solutions provided by nature however is hindered by routine choice of materials. To this end recent years have witnessed a great effort to engineer mechanically flexible photonic devices using polymer substrates. On the other hand, biodegradability and biocompatibility still remains to be incorporated. Hence biomimetics holds the key to overcome the limitations of traditional materials in photonics design. Natural proteins such as sucker ring teeth (SRT) and silk for instance have remarkable mechanical and optical properties that exceed the endeavors of most synthetic and natural polymers. Here we demonstrate for the first time, toroidal whispering gallery mode resonators (WGMR) fabricated entirely from protein structures such as SRT of Loligo vulgaris (European squid) and silk from Bombyx mori. We provide here complete optical and material characterization of proteinaceous WGMRs, revealing high quality factors in microscale and enhancement of Raman signatures by a microcavity. We also present a most simple application of a WGMR as a natural protein add-drop filter, made of SRT protein. Our work shows that with protein-based materials, optical, mechanical and thermal properties can be devised at the molecular level and it lays the groundwork for future eco-friendly, flexible photonics device design.

  11. Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics.

    PubMed

    Allen, S J; Kim, J M; Khorana, H G; Lu, H; Booth, P J

    2001-04-27

    The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to

  12. A thermodynamic and mutational analysis of an RNA purine loop as a protein binding site.

    PubMed

    White, S A; Li, H; Rauch, M E

    1995-10-01

    The thermal stability and protein binding of a 36 nucleotide RNA hairpin containing an internal loop were studied under various solution conditions. Yeast ribosomal protein L32 binds to its transcript and small RNAs which reproduce the L32 transcript's secondary structure have been examined. Replacement of the internal loop with canonical base pairs did not affect the salt dependence of the melting temperature suggesting that both molecules adopt a linear shape. Several electrostatic contacts are formed on binding to a ribosomal fusion protein, but Mg+2 is not required for binding. The RNA protein complex is stable up to 50 degrees C. Two internal loop deletion mutants have similar thermodynamic stabilities and chemical and enzymatic reactivities, but fail to bind the fusion protein. However, several of the internal loop bases of the deletion mutants are moderately reactive to chemical agents whereas the wild type loop sequence displayed a mixed pattern of protection and hyperreactivity.

  13. Flexible non-nucleotide linkers as loop replacements in short double helical RNAs

    PubMed Central

    Pils, Werner; Micura, Ronald

    2000-01-01

    Ethylene glycol oligomers have been studied systematically as non-nucleotide loop replacements in short hairpin oligoribonucleotides. Structural optimization concerns the length of the linkers and is based on the thermodynamic stabilities of the corresponding duplexes. The optimum linker is derived from heptakis (ethylene glycol) provided that the duplex end to be bridged comprises solely the terminal base pair; the optimum linker is derived from hexakis(ethylene glycol) if a dangling unpaired nucleotide is incorporated into the loop. Moreover, these linkers have been compared to other commonly used linker types which consist of repeating units of tris- or tetrakis(ethylene glycol) phosphate, or of 3-hydroxypropane-1-phosphate. In all cases, the correlation between linker length and duplex stability is independent of the kind of counter ions used (Na+, Na+/Mg2+, K+ or Li+). Furthermore, all duplexes with non-nucleotide loop replacements are less stable than those with the corresponding standard nucleotide loop. The results corroborate that the linkers are solvent-exposed and do not specifically interfere with the terminal nucleotides at the bridged duplex end. PMID:10756183

  14. The flexible cytoplasmic loop 3 contributes to the substrate affinity of human monocarboxylate transporters.

    PubMed

    Futagi, Yuya; Sasaki, Shotaro; Kobayashi, Masaki; Narumi, Katsuya; Furugen, Ayako; Iseki, Ken

    2017-10-01

    Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of small molecular weight monocarboxylates. Among hMCTs, hMCT1 exhibits high-affinity l-lactate transport and broad substrate recognition, whereas hMCT4 shows highly specific substrate recognition and low-affinity l-lactate transport, indicating that hMCT1 and hMCT4 have different roles in the body. However, the molecular mechanism of transporter-mediated substrate transport remains unknown. The aim of this study is to identify the domain, which determines the substrate selectivity and affinity of hMCT1 and hMCT4. We constructed a chimera, hMCT4/1, in which the cytoplasmic loop 3 (TM6/7loop) region of hMCT4 was replaced by the corresponding region of hMCT1. Xenopus laevis oocyte heterologous expression system was used to characterize functional features of the chimera. We have demonstrated that the substrate affinity of hMCT1 and hMCT4 depends on the TM6/7loop. Non-conserved His237 residue in the TM6/7loop functions as a regulatory moiety of the substrate affinity. In contrast, the substrate selectivity of the transporters did not depend on the TM6/7loop, suggesting that the domain is not directly involved in substrate recognition. Our study provides important insights into the structures and functions of hMCT1 and hMCT4 transporters. These findings contribute to the development of novel hMCT1 and/or hMCT4 inhibitors as anticancer agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Exploiting protein flexibility to predict the location of allosteric sites

    PubMed Central

    2012-01-01

    Background Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity), by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing that this simple coarse

  16. Falcon: a highly flexible open-source software for closed-loop neuroscience

    NASA Astrophysics Data System (ADS)

    Ciliberti, Davide; Kloosterman, Fabian

    2017-08-01

    Objective. Closed-loop experiments provide unique insights into brain dynamics and function. To facilitate a wide range of closed-loop experiments, we created an open-source software platform that enables high-performance real-time processing of streaming experimental data. Approach. We wrote Falcon, a C++ multi-threaded software in which the user can load and execute an arbitrary processing graph. Each node of a Falcon graph is mapped to a single thread and nodes communicate with each other through thread-safe buffers. The framework allows for easy implementation of new processing nodes and data types. Falcon was tested both on a 32-core and a 4-core workstation. Streaming data was read from either a commercial acquisition system (Neuralynx) or the open-source Open Ephys hardware, while closed-loop TTL pulses were generated with a USB module for digital output. We characterized the round-trip latency of our Falcon-based closed-loop system, as well as the specific latency contribution of the software architecture, by testing processing graphs with up to 32 parallel pipelines and eight serial stages. We finally deployed Falcon in a task of real-time detection of population bursts recorded live from the hippocampus of a freely moving rat. Main results. On Neuralynx hardware, round-trip latency was well below 1 ms and stable for at least 1 h, while on Open Ephys hardware latencies were below 15 ms. The latency contribution of the software was below 0.5 ms. Round-trip and software latencies were similar on both 32- and 4-core workstations. Falcon was used successfully to detect population bursts online with ~40 ms average latency. Significance. Falcon is a novel open-source software for closed-loop neuroscience. It has sub-millisecond intrinsic latency and gives the experimenter direct control of CPU resources. We envisage Falcon to be a useful tool to the neuroscientific community for implementing a wide variety of closed-loop experiments, including those

  17. Falcon: a highly flexible open-source software for closed-loop neuroscience.

    PubMed

    Ciliberti, Davide; Kloosterman, Fabian

    2017-05-26

    Closed-loop experiments provide unique insights into brain dynamics and function. To facilitate a wide range of closed-loop experiments, we created an open-source software platform that enables high-performance real-time processing of streaming experimental data. We wrote Falcon, a C++ multi-threaded software in which the user can load and execute an arbitrary processing graph. Each node of a Falcon graph is mapped to a single thread and nodes communicate with each other through thread-safe buffers. The framework allows for easy implementation of new processing nodes and data types. Falcon was tested both on a 32-core and a 4-core workstation. Streaming data was read from either a commercial acquisition system (Neuralynx) or the open-source Open Ephys hardware, while closed-loop TTL pulses were generated with a USB module for digital output. We characterized the round-trip latency of our Falcon-based closed-loop system, as well as the specific latency contribution of the software architecture, by testing processing graphs with up to 32 parallel pipelines and eight serial stages. We finally deployed Falcon in a task of real-time detection of population bursts recorded live from the hippocampus of a freely moving rat. On Neuralynx hardware, round-trip latency was well below 1 ms and stable for at least 1 h, while on Open Ephys hardware latencies were below 15 ms. The latency contribution of the software was below 0.5 ms. Round-trip and software latencies were similar on both 32- and 4-core workstations. Falcon was used successfully to detect population bursts online with ~40 ms average latency. Falcon is a novel open-source software for closed-loop neuroscience. It has sub-millisecond intrinsic latency and gives the experimenter direct control of CPU resources. We envisage Falcon to be a useful tool to the neuroscientific community for implementing a wide variety of closed-loop experiments, including those requiring use of complex data structures and real

  18. Flexible structural protein alignment by a sequence of local transformations

    PubMed Central

    Rocha, Jairo; Segura, Joan; Wilson, Richard C.; Dasgupta, Swagata

    2009-01-01

    Motivation: Throughout evolution, homologous proteins have common regions that stay semi-rigid relative to each other and other parts that vary in a more noticeable way. In order to compare the increasing number of structures in the PDB, flexible geometrical alignments are needed, that are reliable and easy to use. Results: We present a protein structure alignment method whose main feature is the ability to consider different rigid transformations at different sites, allowing for deformations beyond a global rigid transformation. The performance of the method is comparable with that of the best ones from 10 aligners tested, regarding both the quality of the alignments with respect to hand curated ones, and the classification ability. An analysis of some structure pairs from the literature that need to be matched in a flexible fashion are shown. The use of a series of local transformations can be exported to other classifiers, and a future golden protein similarity measure could benefit from it. Availability: A public server for the program is available at http://dmi.uib.es/ProtDeform/. Contact: jairo@uib.es Supplementary information: All data used, results and examples are available at http://dmi.uib.es/people/jairo/bio/ProtDeform.Supplementary data are available at Bioinformatics online. PMID:19417057

  19. Evaluating the potential of a loop-extended scorpion toxin-like peptide as a protein scaffold.

    PubMed

    Zhang, Shangfei; Zhu, Limei; Yu, Jie; Xu, Jun; Gao, Bin; Zhou, Changlin; Zhu, Shunyi

    2016-12-01

    Grafting of exogenous bioactive sites or functional motifs onto structurally stable scaffolds to gain new functions represents an important research direction in protein engineering. Some engineered proteins have been developed into therapeutic drugs. MeuNaTxα-3 (abbreviated as MT-3) is a newly characterized scorpion sodium channel toxin-like peptide isolated from the venom of the scorpion Mesobuthus eupeus, which contains a rigid scaffold highly similar to classical scorpion sodium channel toxins and an extension of eight amino acids in its J-loop region. This extended loop constitutes a flexible region extruded from the scaffold and could be substituted by exogenous functional sequences. In this study, we experimentally evaluated the scaffold potential of MT-3 through grafting two small antimicrobial motifs to replace residues within the loop. Functional assays showed that the two engineered molecules exhibited elevated antimicrobial potency, as compared with the unmodified scaffold, without structural disruption, providing experimental evidence in favor of MT-3 as a promising scaffold in protein engineering. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Assessing the effect of dynamics on the closed-loop protein-folding hypothesis

    PubMed Central

    Chintapalli, Sree V.; Illingworth, Christopher J. R.; Upton, Graham J. G.; Sacquin-Mora, Sophie; Reeves, Philip J.; Mohammedali, Hani S.; Reynolds, Christopher A.

    2014-01-01

    The closed-loop (loop-n-lock) hypothesis of protein folding suggests that loops of about 25 residues, closed through interactions between the loop ends (locks), play an important role in protein structure. Coarse-grain elastic network simulations, and examination of loop lengths in a diverse set of proteins, each supports a bias towards loops of close to 25 residues in length between residues of high stability. Previous studies have established a correlation between total contact distance (TCD), a metric of sequence distances between contacting residues (cf. contact order), and the log-folding rate of a protein. In a set of 43 proteins, we identify an improved correlation (r2 = 0.76), when the metric is restricted to residues contacting the locks, compared to the equivalent result when all residues are considered (r2 = 0.65). This provides qualified support for the hypothesis, albeit with an increased emphasis upon the importance of a much larger set of residues surrounding the locks. Evidence of a similar-sized protein core/extended nucleus (with significant overlap) was obtained from TCD calculations in which residues were successively eliminated according to their hydrophobicity and connectivity, and from molecular dynamics simulations. Our results suggest that while folding is determined by a subset of residues that can be predicted by application of the closed-loop hypothesis, the original hypothesis is too simplistic; efficient protein folding is dependent on a considerably larger subset of residues than those involved in lock formation. PMID:24258160

  1. Identification of an autoinhibitory region in the activation loop of the Mos protein kinase.

    PubMed Central

    Robertson, S C; Donoghue, D J

    1996-01-01

    The Mos protein is a serine/threonine protein kinase which acts to regulate progression through meiosis in vertebrate oocytes. Although Mos function is dependent on its ability to act as a protein kinase, little is known about the factors which regulate Mos kinase activity. To understand the mechanism by which Mos kinase activity is regulated, we have used molecular modeling to construct a three-dimensional model of Mos based on the crystallographic coordinates of cyclic AMP-dependent kinase (PKA). This model identified a loop in Mos which is positioned near the active site and appears capable of blocking substrate access to the active site. Mutagenesis was used to construct altered forms of the Mos protein with deletions of parts or all of the loop. In vitro kinase assays showed that Mos proteins with the loop removed had up to a fourfold increase in kinase activity compared with the wild-type protein, indicating that the loop acts in an autoinhibitory manner for Mos kinase activity. Point mutations were also made on individual residues of the loop which were determined from the molecular model to be capable of reaching the active site. Determination of the kinase activities of these mutants showed that individual mutations in the loop region are capable of either increasing or decreasing kinase activity with regard to the wild-type protein. These data suggest that the loop identified in Mos acts as an autoinhibitor of kinase activity. PMID:8668163

  2. Synthesis and evaluation of novel 3-C-alkylated-Neu5Ac2en derivatives as probes of influenza virus sialidase 150-loop flexibility.

    PubMed

    Rudrawar, Santosh; Kerry, Philip S; Rameix-Welti, Marie-Anne; Maggioni, Andrea; Dyason, Jeffrey C; Rose, Faith J; van der Werf, Sylvie; Thomson, Robin J; Naffakh, Nadia; Russell, Rupert J M; von Itzstein, Mark

    2012-11-21

    Novel 3-C-alkylated-Neu5Ac2en derivatives have been designed to target the expanded active site cavity of influenza virus sialidases with an open 150-loop, currently seen in X-ray crystal structures of influenza A virus group-1 (N1, N4, N5, N8), but not group-2 (N2, N9), sialidases. The compounds show selectivity for inhibition of H5N1 and pdm09 H1N1 sialidases over an N2 sialidase, providing evidence of the relative 150-loop flexibility of these sialidases. In a complex with N8 sialidase, the C3 substituent of 3-phenylally-Neu5Ac2en occupies the 150-cavity while the central ring and the remaining substituents bind the active site as seen for the unsubstituted template. This new class of inhibitors, which can 'trap' the open 150-loop form of the sialidase, should prove useful as probes of 150-loop flexibility.

  3. Flexibility and small pockets at protein–protein interfaces: New insights into druggability

    PubMed Central

    Jubb, Harry; Blundell, Tom L.; Ascher, David B.

    2015-01-01

    The transient assembly of multiprotein complexes mediates many aspects of cell regulation and signalling in living organisms. Modulation of the formation of these complexes through targeting protein–protein interfaces can offer greater selectivity than the inhibition of protein kinases, proteases or other post-translational regulatory enzymes using substrate, co-factor or transition state mimetics. However, capitalising on protein–protein interaction interfaces as drug targets has been hindered by the nature of interfaces that tend to offer binding sites lacking the well-defined large cavities of classical drug targets. In this review we posit that interfaces formed by concerted folding and binding (disorder-to-order transitions on binding) of one partner and other examples of interfaces where a protein partner is bound through a continuous epitope from a surface-exposed helix, flexible loop or chain extension may be more tractable for the development of “orthosteric”, competitive chemical modulators; these interfaces tend to offer small-volume but deep pockets and/or larger grooves that may be bound tightly by small chemical entities. We discuss examples of such protein–protein interaction interfaces for which successful chemical modulators are being developed. PMID:25662442

  4. Fast De Novo Discovery of Low-energy Protein Loop Conformations.

    PubMed

    Wong, Samuel W K; Liu, Jun S; Kou, S C

    2017-04-05

    In the prediction of protein structure from amino acid sequence, loops are challenging regions for computational methods. Since loops are often located on the protein surface, they can have significant roles in determining protein functions and binding properties. Loop prediction without the aid of a structural template requires extensive conformational sampling and energy minimization, which are computationally difficult. In this article we present a new de novo loop sampling method, the Parallely-filtered Energy Targeted All-atom Loop Sampler (PETALS) to rapidly locate low energy conformations. PETALS explores both backbone and side-chain positions of the loop region simultaneously according to the energy function selected by the user, and constructs a non-redundant ensemble of low energy loop conformations using filtering criteria. The method is illustrated with the DFIRE potential and DiSGro energy function for loops, and shown to be highly effective at discovering conformations with near-native (or better) energy. Using the same energy function as the DiSGro algorithm, PETALS samples conformations with both lower RMSDs and lower energies. PETALS is also useful for assessing the accuracy of different energy functions. PETALS runs rapidly, requiring an average time cost of 10 minutes for a length 12 loop on a single 3.2GHz processor core, comparable to the fastest existing de novo methods for generating an ensemble of conformations. This article is protected by copyright. All rights reserved.

  5. Coupled rotor-flexible fuselage vibration reduction using open loop higher harmonic control

    NASA Technical Reports Server (NTRS)

    Papavassiliou, I.; Friedmann, P. P.; Venkatesan, C.

    1991-01-01

    A fundamental study of vibration prediction and vibration reduction in helicopters using active controls was performed. The nonlinear equations of motion for a coupled rotor/flexible fuselage system have been derived using computer algebra on a special purpose symbolic computer facility. The trim state and vibratory response of the helicopter are obtained in a single pass by applying the harmonic balance technique and simultaneously satisfying the trim and the vibratory response of the helicopter for all rotor and fuselage degrees of freedom. The influence of the fuselage flexibility on the vibratory response is studied. It is shown that the conventional single frequency higher harmonic control is capable of reducing either the hub loads or only the fuselage vibrations but not both simultaneously. It is demonstrated that for simultaneous reduction of hub shears and fuselae vibrations a new scheme called multiple higher harmonic control is required.

  6. Closed-Loop Control Techniques for Active Vibration Suppression of a Flexible Mechanical System

    NASA Astrophysics Data System (ADS)

    Villaverde Huertas, Vladímir; Rohaľ-Ilkiv, Boris

    2012-12-01

    This paper investigates the problem of vibration attenuation of a lightly damped mechanical system using piezoelectric actuation. First of all, an explicit predictive controller will be designed using the Matlab multi-parametric toolbox. Then, we will explore the positive position feedback technique and test the discrete-time PPF controller using an xPC target real-time system. On the other hand, we will realize the modal analysis of the analyzed flexible system in order to determine the frequency corresponding to the first mode shape. This frequency will be utilized as PPF controller frequency. Moreover, the state-space model of the flexible mechanical system will be obtained using the Matlab system identification toolbox applying the subspace identification approach.

  7. Coupled rotor-flexible fuselage vibration reduction using open loop higher harmonic control

    NASA Technical Reports Server (NTRS)

    Papavassiliou, I.; Friedmann, P. P.; Venkatesan, C.

    1991-01-01

    A fundamental study of vibration prediction and vibration reduction in helicopters using active controls was performed. The nonlinear equations of motion for a coupled rotor/flexible fuselage system have been derived using computer algebra on a special purpose symbolic computer facility. The trim state and vibratory response of the helicopter are obtained in a single pass by applying the harmonic balance technique and simultaneously satisfying the trim and the vibratory response of the helicopter for all rotor and fuselage degrees of freedom. The influence of the fuselage flexibility on the vibratory response is studied. It is shown that the conventional single frequency higher harmonic control is capable of reducing either the hub loads or only the fuselage vibrations but not both simultaneously. It is demonstrated that for simultaneous reduction of hub shears and fuselae vibrations a new scheme called multiple higher harmonic control is required.

  8. A Structure-free Method for Quantifying Conformational Flexibility in proteins

    PubMed Central

    Burger, Virginia M.; Arenas, Daniel J.; Stultz, Collin M.

    2016-01-01

    All proteins sample a range of conformations at physiologic temperatures and this inherent flexibility enables them to carry out their prescribed functions. A comprehensive understanding of protein function therefore entails a characterization of protein flexibility. Here we describe a novel approach for quantifying a protein’s flexibility in solution using small-angle X-ray scattering (SAXS) data. The method calculates an effective entropy that quantifies the diversity of radii of gyration that a protein can adopt in solution and does not require the explicit generation of structural ensembles to garner insights into protein flexibility. Application of this structure-free approach to over 200 experimental datasets demonstrates that the methodology can quantify a protein’s disorder as well as the effects of ligand binding on protein flexibility. Such quantitative descriptions of protein flexibility form the basis of a rigorous taxonomy for the description and classification of protein structure. PMID:27358108

  9. Hinge Atlas: relating protein sequence to sites of structural flexibility

    PubMed Central

    Flores, Samuel C; Lu, Long J; Yang, Julie; Carriero, Nicholas; Gerstein, Mark B

    2007-01-01

    Background Relating features of protein sequences to structural hinges is important for identifying domain boundaries, understanding structure-function relationships, and designing flexibility into proteins. Efforts in this field have been hampered by the lack of a proper dataset for studying characteristics of hinges. Results Using the Molecular Motions Database we have created a Hinge Atlas of manually annotated hinges and a statistical formalism for calculating the enrichment of various types of residues in these hinges. Conclusion We found various correlations between hinges and sequence features. Some of these are expected; for instance, we found that hinges tend to occur on the surface and in coils and turns and to be enriched with small and hydrophilic residues. Others are less obvious and intuitive. In particular, we found that hinges tend to coincide with active sites, but unlike the latter they are not at all conserved in evolution. We evaluate the potential for hinge prediction based on sequence. Motions play an important role in catalysis and protein-ligand interactions. Hinge bending motions comprise the largest class of known motions. Therefore it is important to relate the hinge location to sequence features such as residue type, physicochemical class, secondary structure, solvent exposure, evolutionary conservation, and proximity to active sites. To do this, we first generated the Hinge Atlas, a set of protein motions with the hinge locations manually annotated, and then studied the coincidence of these features with the hinge location. We found that all of the features have bearing on the hinge location. Most interestingly, we found that hinges tend to occur at or near active sites and yet unlike the latter are not conserved. Less surprisingly, we found that hinge residues tend to be small, not hydrophobic or aliphatic, and occur in turns and random coils on the surface. A functional sequence based hinge predictor was made which uses some of the

  10. Protein flexibility acclimatizes photosynthetic energy conversion to the ambient temperature.

    PubMed

    Shlyk-Kerner, Oksana; Samish, Ilan; Kaftan, David; Holland, Neta; Sai, P S Maruthi; Kless, Hadar; Scherz, Avigdor

    2006-08-17

    Adjustment of catalytic activity in response to diverse ambient temperatures is fundamental to life on Earth. A crucial example of this is photosynthesis, where solar energy is converted into electrochemical potential that drives oxygen and biomass generation at temperatures ranging from those of frigid Antarctica to those of scalding hot springs. The energy conversion proceeds by concerted mobilization of electrons and protons on photoexcitation of reaction centre protein complexes. Following physicochemical paradigms, the rates of imperative steps in this process were predicted to increase exponentially with rising temperatures, resulting in different yields of solar energy conversion at the distinct growth temperatures of photosynthetic mesophiles and extremophiles. In contrast, here we show a meticulous adjustment of energy conversion rate, resulting in similar yields from mesophiles and thermophiles. The key molecular players in the temperature adjustment process consist of a cluster of hitherto unrecognized protein cavities and an adjacent packing motif that jointly impart local flexibility crucial to the reaction centre proteins. Mutations within the packing motif of mesophiles that increase the bulkiness of the amino-acid side chains, and thus reduce the size of the cavities, promote thermophilic behaviour. This novel biomechanical mechanism accounts for the slowing of the catalytic reaction above physiological temperatures in contradiction to the classical Arrhenius paradigm. The mechanism provides new guidelines for manipulating the acclimatization of enzymes to the ambient temperatures of diverse habitats. More generally, it reveals novel protein elements that are of potential significance for modulating structure-activity relationships in membrane and globular proteins alike.

  11. Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility

    PubMed Central

    Loving, Kathryn A.; Lin, Andy; Cheng, Alan C.

    2014-01-01

    Advances reported over the last few years and the increasing availability of protein crystal structure data have greatly improved structure-based druggability approaches. However, in practice, nearly all druggability estimation methods are applied to protein crystal structures as rigid proteins, with protein flexibility often not directly addressed. The inclusion of protein flexibility is important in correctly identifying the druggability of pockets that would be missed by methods based solely on the rigid crystal structure. These include cryptic pockets and flexible pockets often found at protein-protein interaction interfaces. Here, we apply an approach that uses protein modeling in concert with druggability estimation to account for light protein backbone movement and protein side-chain flexibility in protein binding sites. We assess the advantages and limitations of this approach on widely-used protein druggability sets. Applying the approach to all mammalian protein crystal structures in the PDB results in identification of 69 proteins with potential druggable cryptic pockets. PMID:25079060

  12. Structure-based druggability assessment of the mammalian structural proteome with inclusion of light protein flexibility.

    PubMed

    Loving, Kathryn A; Lin, Andy; Cheng, Alan C

    2014-07-01

    Advances reported over the last few years and the increasing availability of protein crystal structure data have greatly improved structure-based druggability approaches. However, in practice, nearly all druggability estimation methods are applied to protein crystal structures as rigid proteins, with protein flexibility often not directly addressed. The inclusion of protein flexibility is important in correctly identifying the druggability of pockets that would be missed by methods based solely on the rigid crystal structure. These include cryptic pockets and flexible pockets often found at protein-protein interaction interfaces. Here, we apply an approach that uses protein modeling in concert with druggability estimation to account for light protein backbone movement and protein side-chain flexibility in protein binding sites. We assess the advantages and limitations of this approach on widely-used protein druggability sets. Applying the approach to all mammalian protein crystal structures in the PDB results in identification of 69 proteins with potential druggable cryptic pockets.

  13. A flexible loop-type flow modulator for comprehensive two-dimensional gas chromatography.

    PubMed

    Tranchida, Peter Quinto; Purcaro, Giorgia; Visco, Alessandro; Conte, Lanfranco; Dugo, Paola; Dawes, Peter; Mondello, Luigi

    2011-05-27

    The present investigation is focused on a simple flow modulator (FM), for comprehensive two-dimensional gas chromatography (GC×GC). The interface is stable at high temperatures, and consists of a metallic disc (located inside the GC oven) with seven ports, which are connected to an auxiliary pressure source via two branches, to the first and second dimension, to a waste branch (linked to a needle valve) and to an exchangeable modulation loop (2 ports). The ports are connected via micro-channels, etched on one of the inner surfaces of the disc. Modulation is achieved using a two-way electrovalve, connected on one side to the additional pressure source, and to the two metal branches, on the other. An FM enantio-GC×polar-GC method (using a flame ionization detector) was optimized (a 40-μL loop was employed), for the analysis of essential oils. As an example, an application on spearmint oil is shown; the method herein proposed was subjected to validation. Finally, an FM GC×GC diesel experiment was carried out, using an apolar-polar column combination, to demonstrate the effectiveness of the modulator in the analysis of a totally different sample-type. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. Structural families in loops of homologous proteins: automatic classification, modelling and application to antibodies.

    PubMed

    Martin, A C; Thornton, J M

    1996-11-15

    Loop regions of polypeptide in homologous proteins may be classified into structural families. A method is described by which this classification may be performed automatically and "key residue" templates, which may be responsible for the loop adopting a given conformation, are defined. The technique has been applied to the hypervariable loops of antibodies and the results are compared with the previous definition of canonical classes. We have extended these definitions and provide complete sets of structurally determining residues (SDRs) for the observed clusters including the first set of key residues for seven-residue CDR-H3 loops.

  15. Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping.

    PubMed

    Linask, Kersti K; Han, Ming-Da; Linask, Kaari L; Schlange, Thomas; Brand, Thomas

    2003-10-01

    Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping. Copyright 2003 Wiley-Liss, Inc.

  16. Kinks, loops, and protein folding, with protein A as an example

    PubMed Central

    Krokhotin, Andrey; Liwo, Adam; Maisuradze, Gia G.; Niemi, Antti J.; Scheraga, Harold A.

    2014-01-01

    The dynamics and energetics of formation of loops in the 46-residue N-terminal fragment of the B-domain of staphylococcal protein A has been studied. Numerical simulations have been performed using coarse-grained molecular dynamics with the united-residue (UNRES) force field. The results have been analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger (DNLS) equation. In the case of proteins, the DNLS equation arises from a Cα-trace-based energy function. Three individual kink profiles were identified in the experimental three-α-helix structure of protein A, in the range of the Glu16-Asn29, Leu20-Asn29, and Gln33-Asn44 residues, respectively; these correspond to two loops in the native structure. UNRES simulations were started from the full right-handed α-helix to obtain a clear picture of kink formation, which would otherwise be blurred by helix formation. All three kinks emerged during coarse-grained simulations. It was found that the formation of each is accompanied by a local free energy increase; this is expressed as the change of UNRES energy which has the physical sense of the potential of mean force of a polypeptide chain. The increase is about 7 kcal/mol. This value can thus be considered as the free energy barrier to kink formation in full α-helical segments of polypeptide chains. During the simulations, the kinks emerge, disappear, propagate, and annihilate each other many times. It was found that the formation of a kink is initiated by an abrupt change in the orientation of a pair of consecutive side chains in the loop region. This resembles the formation of a Bloch wall along a spin chain, where the Cα backbone corresponds to the chain, and the amino acid side chains are interpreted as the spin variables. This observation suggests that nearest-neighbor side chain–side chain interactions are responsible for initiation of loop formation. It was also found that the individual kinks are

  17. Kinks, loops, and protein folding, with protein A as an example

    SciTech Connect

    Krokhotin, Andrey; Liwo, Adam; Maisuradze, Gia G. Scheraga, Harold A.; Niemi, Antti J.

    2014-01-14

    The dynamics and energetics of formation of loops in the 46-residue N-terminal fragment of the B-domain of staphylococcal protein A has been studied. Numerical simulations have been performed using coarse-grained molecular dynamics with the united-residue (UNRES) force field. The results have been analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger (DNLS) equation. In the case of proteins, the DNLS equation arises from a C{sup α}-trace-based energy function. Three individual kink profiles were identified in the experimental three-α-helix structure of protein A, in the range of the Glu16-Asn29, Leu20-Asn29, and Gln33-Asn44 residues, respectively; these correspond to two loops in the native structure. UNRES simulations were started from the full right-handed α-helix to obtain a clear picture of kink formation, which would otherwise be blurred by helix formation. All three kinks emerged during coarse-grained simulations. It was found that the formation of each is accompanied by a local free energy increase; this is expressed as the change of UNRES energy which has the physical sense of the potential of mean force of a polypeptide chain. The increase is about 7 kcal/mol. This value can thus be considered as the free energy barrier to kink formation in full α-helical segments of polypeptide chains. During the simulations, the kinks emerge, disappear, propagate, and annihilate each other many times. It was found that the formation of a kink is initiated by an abrupt change in the orientation of a pair of consecutive side chains in the loop region. This resembles the formation of a Bloch wall along a spin chain, where the C{sup α} backbone corresponds to the chain, and the amino acid side chains are interpreted as the spin variables. This observation suggests that nearest-neighbor side chain–side chain interactions are responsible for initiation of loop formation. It was also found that the individual

  18. Flexible Airspace Management (FAM) Research 2010 Human-in-the-Loop Simulation

    NASA Technical Reports Server (NTRS)

    Lee, Paul U.; Brasil, Connie; Homola, Jeffrey; Kessell, Angela; Prevot, Thomas; Smith, Nancy

    2011-01-01

    A human-in-the-Ioop (HITL) simulation was conducted to assess potential user and system benefits of Flexible Airspace Management (FAM) concept, as well as designing role definitions, procedures, and tools to support the FAM operations in the mid-term High Altitude Airspace (HAA) environment. The study evaluated the benefits and feasibility of flexible airspace reconfiguration in response to traffic overload caused by weather deviations, and compared them to those in a baseline condition without the airspace reconfiguration. The test airspace consisted of either four sectors in one Area of Specialization or seven sectors across two Areas. The test airspace was assumed to be at or above FL340 and fully equipped Vvith data communications (Data Comm). Other assumptions were consistent with those of the HAA concept. Overall, results showed that FAM operations with multiple Traffic Management Coordinators, Area Supervisors, and controllers worked remarkably well. The results showed both user and system benefits, some of which include the increased throughput, decreased flight distance, more manageable sector loads, and better utilized airspace. Also, the roles, procedures, airspace designs, and tools were all very well received. Airspace configuration options that resulted from a combination of algorithm-generated airspace configurations with manual modifications were well acceptec and posed little difficuIty and/or workload during airspace reconfiguration process. The results suggest a positive impact of FAM operations in HAA. Further investigation would be needed to evaluate if the benefits and feasibility would extend in either non-HAA or mixed equipage environment.

  19. Protein flexibility oriented virtual screening strategy for JAK2 inhibitors

    NASA Astrophysics Data System (ADS)

    Xiong, Xiao; Yuan, Haoliang; Zhang, Yanmin; Xu, Jinxing; Ran, Ting; Liu, Haichun; Lu, Shuai; Xu, Anyang; Li, Hongmei; Jiang, Yulei; Lu, Tao; Chen, Yadong

    2015-10-01

    JAK2 has been considered as an important target for the development of anti-cancer agents. In this study, considering the flexibility of its binding site, an integrated strategy combining Bayesian categorization modeling and ensemble docking was established. Four representative crystal structures were selected for ensemble docking by the hierarchical clustering of 34 crystal structures according to the volume overlaps of each structure. A retrospective virtual screening was performed to validate this integrated strategy. As the preliminary filtration, the Bayesian model enhanced the ratio of actives by reducing the large amount of decoys. After docking the remaining compounds, the comparison between the ensemble and individual results showed that the enrichment of ensemble docking improved significantly. The results of analysis on conformational changes of two top ranked active inhibitors when docking into different proteins indicated that compounds with flexible conformations well fitted the different binding site shapes were more likely to be potential JAK2 inhibitors. This high efficient strategy will facilitate virtual screening for novel JAK2 inhibitors and could be even applied in drug discovery against other targets.

  20. Probing protein flexibility reveals a mechanism for selective promiscuity

    PubMed Central

    Pabon, Nicolas A; Camacho, Carlos J

    2017-01-01

    Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets. DOI: http://dx.doi.org/10.7554/eLife.22889.001 PMID:28432789

  1. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors

    PubMed Central

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-01-01

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process. PMID:27438863

  2. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

    PubMed

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-07-18

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

  3. Interplay of Protein Binding Interactions, DNA Mechanics, and Entropy in DNA Looping Kinetics

    PubMed Central

    Mulligan, Peter J.; Chen, Yi-Ju; Phillips, Rob; Spakowitz, Andrew J.

    2015-01-01

    DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions. PMID:26244743

  4. A modular perspective of protein structures: application to fragment based loop modeling.

    PubMed

    Fernandez-Fuentes, Narcis; Fiser, Andras

    2013-01-01

    Proteins can be decomposed into supersecondary structure modules. We used a generic definition of supersecondary structure elements, so-called Smotifs, which are composed of two flanking regular secondary structures connected by a loop, to explore the evolution and current variety of structure building blocks. Here, we discuss recent observations about the saturation of Smotif geometries in protein structures and how it opens new avenues in protein structure modeling and design. As a first application of these observations we describe our loop conformation modeling algorithm, ArchPred that takes advantage of Smotifs classification. In this application, instead of focusing on specific loop properties the method narrows down possible template conformations in other, often not homologous structures, by identifying the most likely supersecondary structure environment that cradles the loop. Beyond identifying the correct starting supersecondary structure geometry, it takes into account information of fit of anchor residues, sterical clashes, match of predicted and observed dihedral angle preferences, and local sequence signal.

  5. Control of the conductance of engineered protein nanopores through concerted loop motions.

    PubMed

    Zhuang, Tiandi; Tamm, Lukas K

    2014-06-02

    Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized as the main gating loop that closes the pore at low pH values and opens it at high pH values. A method was devised to pin each of the loops to the embedding membrane and measure the single-pore conductances of the resulting constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighboring and distant loops in a correlated fashion. Pinning loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity.

    PubMed

    Galm, Lara; Amrhein, Sven; Hubbuch, Jürgen

    2016-02-08

    The aggregation of proteins became one of the major challenges in the development of biopharmaceu-ticals since the formation of aggregates can affect drug quality and immunogenicity. However, aggregation mechanisms are highly complex and the investigation requires cost, time, and material intensive experi-mental effort. In the present work, the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied. In particular, the surface hydrophobicity, zeta potential, and conformational flexibility of human lysozyme, lysozyme from chicken egg white, and α-lactalbumin at pH 3, 5, 7, and 9 were assessed and examined for correlation with experimental stability studies focusing on protein phase behavior induced by sodium chloride and ammonium sulfate. The molecular dynamics (MD) simulation based study of the conformational flexibility without precipitants was able to identify highly flexible protein regions which could be associated to the less regular secondary structure elements and random coiled and terminal regions in particular. Conformational flex-ibility of the entire protein structure and protein surface hydrophobicity could be correlated to differing aggregation propensities among the studied proteins and could be identified to be applicable for predic-tion of protein phase behavior in aqueous solution without precipitants. For prediction of protein phase behavior and aggregation propensity in aqueous solution with precipitants, protein flexibility was further studied in dependency of salt concentration and species by means of human lysozyme. Even though the results of the salt dependent MD simulations could not be shown to be sufficient for prediction of salt depending phase behavior, this study revealed a more pronounced destabilizing effect of ammonium sulfate in comparison to sodium chloride and thus, was found to be in good agreement with theoretical considerations

  7. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    NASA Astrophysics Data System (ADS)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  8. Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

    PubMed Central

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J.

    2012-01-01

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens. PMID:22646091

  9. Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

    SciTech Connect

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J.

    2012-11-01

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k{sub cat}. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

  10. 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}).

  11. The role of thermal fluctuations and mechanical constraints in protein-mediated DNA looping

    NASA Astrophysics Data System (ADS)

    Blumberg, Seth; Gajraj, Arivalagan; Pennington, Matthew; Tkachenko, Alexei; Meiners, Jens-Christian

    2005-05-01

    Protein-mediated DNA looping, which occurs when a linker protein binds to two operator sites on the same DNA molecule, is an important regulatory element of many biological processes such as transcription and DNA replication. In physiologic conditions, the conformation of DNA undergoes thermal fluctuations which enable the operators to align for looping. The likelihood for the operator sites to align can be significantly altered by mechanically constraining the substrate DNA. For instance, tension extends DNA and increases the free energy of operator alignment. By modeling DNA as a wormlike chain, we use statistical mechanics to show that when the loop size is greater than 100bp a tension of 500 femtonewtons can increase the time required for loop closure by two orders of magnitude. This force is small compared to the piconewton forces that are associated with RNA polymerases and other molecular motors, indicating that intracellular mechanical forces might affect transcriptional regulation. We propose that supercoiling of DNA may help to stabilize the looping process against the disruptive effective of tension. Since DNA looping is important in gene regulation and genetic transformation, our theory suggests that thermal fluctuations and response to mechanical constraints play an important role in a living cell. Indeed, recent micromechanical measurements on DNA looping have verified the importance of mechanical constraints. Besides providing perspective on these experiments we offer suggestions for future micromechanical studies.

  12. Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus.

    PubMed

    Yang, Shaoqing; Wang, Tao; Bohon, Jen; Gagné, Marie-Ève Laliberté; Bolduc, Marilène; Leclerc, Denis; Li, Huilin

    2012-09-14

    Papaya mosaic virus (PapMV) is a filamentous plant virus that belongs to the Alphaflexiviridae family. Flexible filamentous viruses have defied more than two decades of effort in fiber diffraction, and no high-resolution structure is available for any member of the Alphaflexiviridae family. Here, we report our structural characterization of PapMV by X-ray crystallography and cryo-electron microscopy three-dimensional reconstruction. We found that PapMV is 135Å in diameter with a helical symmetry of ~10 subunits per turn. Crystal structure of the C-terminal truncated PapMV coat protein (CP) reveals a novel all-helix fold with seven α-helices. Thus, the PapMVCP structure is different from the four-helix-bundle fold of tobacco mosaic virus in which helix bundling dominates the subunit interface in tobacco mosaic virus and conveys rigidity to the rod virus. PapMV CP was crystallized as an asymmetrical dimer in which one protein lassoes the other by the N-terminal peptide. Mutation of residues critical to the inter-subunit lasso interaction abolishes CP polymerization. The crystal structure suggests that PapMV may polymerize via the consecutive N-terminal loop lassoing mechanism. The structure of PapMV will be useful for rational design and engineering of the PapMV nanoparticles into innovative vaccines.

  13. A Continuous-Adaptive DDRx Interface with Flexible Round-Trip-Time and Full Self Loop-Backed AC Test

    NASA Astrophysics Data System (ADS)

    Haraguchi, Masaru; Osawa, Tokuya; Yamazaki, Akira; Morishima, Chikayoshi; Morihara, Toshinori; Morooka, Yoshikazu; Okuno, Yoshihiro; Arimoto, Kazutami

    This paper describes new DDRx SDRAM interface architecture suitable for system-on-chip (SOC) implementation. Our test chip fabricated in a 90-nm CMOS process adopts three key schemes and achieves 960 Mb/s/pin operations with 32 bits width. One of new schemes is to suppress timing skew with rising-edge signal transmission I/O circuit and look-up table type impedance calibration circuit. DQS round-trip-time, propagation delay from rising edge of system clock in SOC to arrival of DQS at input PAD of SOC during read operation, becomes longer than one clock cycle time as for DDR2 interface and beyond. Flexible DQS round-trip-time scheme can allow wide range up to N/2 cycles in N bits burst read operation. In addition, full self loop-backed test scheme is also proposed to measure AC timing parameters without high-end tester. The architecture reported in this paper can be continuously adaptive to realize higher data-rate and cost-efficient DDRx-SDRAM interface for various kinds of SOC.

  14. Structural flexibility of intrinsically disordered proteins induces stepwise target recognition.

    PubMed

    Shirai, Nobu C; Kikuchi, Macoto

    2013-12-14

    An intrinsically disordered protein (IDP) lacks a stable three-dimensional structure, while it folds into a specific structure when it binds to a target molecule. In some IDP-target complexes, not all target binding surfaces are exposed on the outside, and intermediate states are observed in their binding processes. We consider that stepwise target recognition via intermediate states is a characteristic of IDP binding to targets with "hidden" binding sites. To investigate IDP binding to hidden target binding sites, we constructed an IDP lattice model based on the HP model. In our model, the IDP is modeled as a chain and the target is modeled as a highly coarse-grained object. We introduced motion and internal interactions to the target to hide its binding sites. In the case of unhidden binding sites, a two-state transition between the free states and a bound state is observed, and we consider that this represents coupled folding and binding. Introducing hidden binding sites, we found an intermediate bound state in which the IDP forms various structures to temporarily stabilize the complex. The intermediate state provides a scaffold for the IDP to access the hidden binding site. We call this process multiform binding. We conclude that structural flexibility of IDPs enables them to access hidden binding sites and this is a functional advantage of IDPs.

  15. Structural flexibility of intrinsically disordered proteins induces stepwise target recognition

    NASA Astrophysics Data System (ADS)

    Shirai, Nobu C.; Kikuchi, Macoto

    2013-12-01

    An intrinsically disordered protein (IDP) lacks a stable three-dimensional structure, while it folds into a specific structure when it binds to a target molecule. In some IDP-target complexes, not all target binding surfaces are exposed on the outside, and intermediate states are observed in their binding processes. We consider that stepwise target recognition via intermediate states is a characteristic of IDP binding to targets with "hidden" binding sites. To investigate IDP binding to hidden target binding sites, we constructed an IDP lattice model based on the HP model. In our model, the IDP is modeled as a chain and the target is modeled as a highly coarse-grained object. We introduced motion and internal interactions to the target to hide its binding sites. In the case of unhidden binding sites, a two-state transition between the free states and a bound state is observed, and we consider that this represents coupled folding and binding. Introducing hidden binding sites, we found an intermediate bound state in which the IDP forms various structures to temporarily stabilize the complex. The intermediate state provides a scaffold for the IDP to access the hidden binding site. We call this process multiform binding. We conclude that structural flexibility of IDPs enables them to access hidden binding sites and this is a functional advantage of IDPs.

  16. Sampling Multiple Scoring Functions Can Improve Protein Loop Structure Prediction Accuracy

    PubMed Central

    Rata, Ionel; Jakobsson, Eric

    2011-01-01

    Accurately predicting loop structures is important for understanding functions of many proteins. In order to obtain loop models with high accuracy, efficiently sampling the loop conformation space to discover reasonable structures is a critical step. In loop conformation sampling, coarse-grain energy (scoring) functions coupling with reduced protein representations are often used to reduce the number of degrees of freedom as well as sampling computational time. However, due to implicitly considering many factors by reduced representations, the coarse-grain scoring functions may have potential insensitivity and inaccuracy, which can mislead the sampling process and consequently ignore important loop conformations. In this paper, we present a new computational sampling approach to obtain reasonable loop backbone models, so-called the Pareto Optimal Sampling (POS) method. The rationale of the POS method is to sample the function space of multiple, carefully-selected scoring functions to discover an ensemble of diversified structures yielding Pareto optimality to all sampled conformations. POS method can efficiently tolerate insensitivity and inaccuracy in individual scoring functions and thereby lead to significant accuracy improvement in loop structure prediction. We apply the POS method to a set of 4- to 12-residue loop targets using a function space composed of backbone-only Rosetta, DFIRE, and a triplet backbone dihedral potential developed in our lab. Our computational results show that in 501 out of 502 targets, the model sets generated by POS contain structure models are within subangstrom resolution. Moreover, the top-ranked models have Root Mean Square Deviation (RMSD) less than 1A in 96.8%, 84.1%, and 72.2% of the short (4~6 residues), medium (7~9 residues), and long (10~12) targets, respectively, when the all-atom models are generated by local optimization from the backbone models and are ranked by our recently developed Pareto Optimal Consensus (POC

  17. Universality in the Timescales of Internal Loop Formation in Unfolded Proteins and Single-Stranded Oligonucleotides

    PubMed Central

    Cheng, Ryan R.; Uzawa, Takanori; Plaxco, Kevin W.; Makarov, Dmitrii E.

    2010-01-01

    Understanding the rate at which various parts of a molecular chain come together to facilitate the folding of a biopolymer (e.g., a protein or RNA) into its functional form remains an elusive goal. Here we use experiments, simulations, and theory to study the kinetics of internal loop closure in disordered biopolymers such as single-stranded oligonucleotides and unfolded proteins. We present theoretical arguments and computer simulation data to show that the relationship between the timescale of internal loop formation and the positions of the monomers enclosing the loop can be recast in a form of a universal master dependence. We also perform experimental measurements of the loop closure times of single-stranded oligonucleotides and show that both these and previously reported internal loop closure kinetics of unfolded proteins are well described by this theoretically predicted dependence. Finally, we propose that experimental deviations from the master dependence can then be used as a sensitive probe of dynamical and structural order in unfolded proteins and other biopolymers. PMID:21156138

  18. Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

    PubMed Central

    Angus, Allan G. N.; Loquet, Antoine; Stack, Séamus J.; Dalrymple, David; Gatherer, Derek

    2012-01-01

    Hepatitis C virus core protein forms the viral nucleocapsid and plays a critical role in the formation of infectious particles. In this study, we demonstrate that the highly conserved residue G33, located within domain 1 of the core protein, is important for the production of cell culture-infectious virus (HCVcc). Alanine substitution at this position in the JFH1 genome did not alter viral RNA replication but reduced infectivity by ∼2 logs. Virus production by this core mutant could be rescued by compensatory mutations located immediately upstream and downstream of the original G33A mutation. The examination of the helix-loop-helix motif observed in the core protein structure (residues 15 to 41; Protein Data Bank entry 1CWX) indicated that the residues G33 and F24 are in close contact with each other, and that the G33A mutation induces a steric clash with F24. Molecular simulations revealed that the compensatory mutations increase the helix-loop-helix flexibility, allowing rescue of the core active conformation required for efficient virus production. Taken together, these data highlight the plasticity of core domain 1 conformation and illustrate the relationship between its structural tolerance to mutations and virus infectivity. PMID:22072760

  19. Effects of T-loop modification on the PII-signalling protein: structure of uridylylated Escherichia coli GlnB bound to ATP.

    PubMed

    Palanca, Carles; Rubio, Vicente

    2017-03-26

    To adapt to environments with variable nitrogen sources and richness, the widely distributed homotrimeric PII signalling proteins bind their allosteric effectors ADP/ATP/2-oxoglutarate, and experience nitrogen-sensitive uridylylation of their flexible T-loops at Tyr51, regulating their interactions with effector proteins. To clarify whether uridylylation triggers a given T-loop conformation, we determined the crystal structure of the classical paradigm of PII protein, Escherichia coli GlnB (EcGlnB), in fully uridylylated form (EcGlnB-UMP3 ). This is the first structure of a postranslationally modified PII protein. This required recombinant production and purification of the uridylylating enzyme GlnD and its use for full uridylylation of large amounts of recombinantly produced pure EcGlnB. Unlike crystalline non-uridylylated EcGlnB, in which T-loops are fixed, uridylylation rendered the T-loop highly mobile because of loss of contacts mediated by Tyr51, with concomitant abolition of T-loop anchoring via Arg38 on the ATP site. This site was occupied by ATP, providing the first, long-sought snapshot of the EcGlnB-ATP complex, connecting ATP binding with T-loop changes. Inferences are made on the mechanisms of PII selectivity for ATP and of PII-UMP3 signalling, proposing a model for the architecture of the complex of EcGlnB-UMP3 with the uridylylation-sensitive PII target ATase (which adenylylates/deadenylylates glutamine synthetase [GS]) and with GS.

  20. Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases*

    PubMed Central

    Bu, Lintao; Crowley, Michael F.; Himmel, Michael E.; Beckham, Gregg T.

    2013-01-01

    Cellulase enzymes cleave glycosidic bonds in cellulose to produce cellobiose via either retaining or inverting hydrolysis mechanisms, which are significantly pH-dependent. Many fungal cellulases function optimally at pH ∼5, and their activities decrease dramatically at higher or lower pH. To understand the molecular-level implications of pH in cellulase structure, we use a hybrid, solvent-based, constant pH molecular dynamics method combined with pH-based replica exchange to determine the pKa values of titratable residues of a glycoside hydrolase (GH) family 6 cellobiohydrolase (Cel6A) and a GH family 7 cellobiohydrolase (Cel7A) from the fungus Hypocrea jecorina. For both enzymes, we demonstrate that a bound substrate significantly affects the pKa values of the acid residues at the catalytic center. The calculated pKa values of catalytic residues confirm their proposed roles from structural studies and are consistent with the experimentally measured apparent pKa values. Additionally, GHs are known to impart a strained pucker conformation in carbohydrate substrates in active sites for catalysis, and results from free energy calculations combined with constant pH molecular dynamics suggest that the correct ring pucker is stable near the optimal pH for both Cel6A and Cel7A. Much longer molecular dynamics simulations of Cel6A and Cel7A with fixed protonation states based on the calculated pKa values suggest that pH affects the flexibility of tunnel loops, which likely affects processivity and substrate complexation. Taken together, this work demonstrates several molecular-level effects of pH on GH enzymes important for cellulose turnover in the biosphere and relevant to biomass conversion processes. PMID:23504310

  1. Prediction of Long Loops with Embedded Secondary Structure using the Protein Local Optimization Program

    PubMed Central

    Miller, Edward B.; Murrett, Colleen S.; Zhu, Kai; Zhao, Suwen; Goldfeld, Dahlia A.; Bylund, Joseph H.; Friesner, Richard A.

    2013-01-01

    Robust homology modeling to atomic-level accuracy requires in the general case successful prediction of protein loops containing small segments of secondary structure. Further, as loop prediction advances to success with larger loops, the exclusion of loops containing secondary structure becomes awkward. Here, we extend the applicability of the Protein Local Optimization Program (PLOP) to loops up to 17 residues in length that contain either helical or hairpin segments. In general, PLOP hierarchically samples conformational space and ranks candidate loops with a high-quality molecular mechanics force field. For loops identified to possess α-helical segments, we employ an alternative dihedral library composed of (ϕ,ψ) angles commonly found in helices. The alternative library is searched over a user-specified range of residues that define the helical bounds. The source of these helical bounds can be from popular secondary structure prediction software or from analysis of past loop predictions where a propensity to form a helix is observed. Due to the maturity of our energy model, the lowest energy loop across all experiments can be selected with an accuracy of sub-Ångström RMSD in 80% of cases, 1.0 to 1.5 Å RMSD in 14% of cases, and poorer than 1.5 Å RMSD in 6% of cases. The effectiveness of our current methods in predicting hairpin-containing loops is explored with hairpins up to 13 residues in length and again reaching an accuracy of sub-Ångström RMSD in 83% of cases, 1.0 to 1.5 Å RMSD in 10% of cases, and poorer than 1.5 Å RMSD in 7% of cases. Finally, we explore the effect of an imprecise surrounding environment, in which side chains, but not the backbone, are initially in perturbed geometries. In these cases, loops perturbed to 3Å RMSD from the native environment were restored to their native conformation with sub-Ångström RMSD. PMID:23814507

  2. Prediction of Loops in G Protein-Coupled Receptor Homology Models: Effect of Imprecise Surroundings and Constraints.

    PubMed

    Arora, Bhumika; Coudrat, Thomas; Wootten, Denise; Christopoulos, Arthur; Noronha, Santosh B; Sexton, Patrick M

    2016-04-25

    In the present study, we explored the extent to which inaccuracies inherent in homology models of the transmembrane helical cores of G protein-coupled receptors (GPCRs) can impact loop prediction. We demonstrate that loop prediction in homology models is much more difficult than loop reconstruction in crystal structures because of the imprecise positioning of loop anchors. Deriving information from 17 recently available GPCR crystal structures, we estimated all of the possible errors that could occur in loop anchors as the result of comparative modeling. Subsequently, we performed an exhaustive analysis to decipher the effect of these errors on loop modeling using ICM High Precision Sampling. The influence of the presence of other extracellular loops was also explored. Our results reveal that the error space of modeled loop residues is much larger than that of the anchor residues, although modeling a particular extracellular loop in the presence of other extracellular loops provides constraints that help in predicting near-native loop conformations observed in crystal structures. This implies that errors in loop anchor positions introduce increased uncertainty in the modeled loop coordinates. Therefore, for the success of any GPCR structure prediction algorithm, minimizing errors in the helical end points is likely to be critical for successful loop modeling.

  3. Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins*

    PubMed Central

    Bondos, Sarah E.; Swint-Kruse, Liskin; Matthews, Kathleen S.

    2015-01-01

    To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: the prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection. PMID:26342073

  4. Sampling the conformation of protein surface residues for flexible protein docking

    PubMed Central

    2010-01-01

    Background The problem of determining the physical conformation of a protein dimer, given the structures of the two interacting proteins in their unbound state, is a difficult one. The location of the docking interface is determined largely by geometric complementarity, but finding complementary geometry is complicated by the flexibility of the backbone and side-chains of both proteins. We seek to generate candidates for docking that approximate the bound state well, even in cases where there is backbone and/or side-chain difference from unbound to bound states. Results We divide the surfaces of each protein into local patches and describe the effect of side-chain flexibility on each patch by sampling the space of conformations of its side-chains. Likely positions of individual side-chains are given by a rotamer library; this library is used to derive a sample of possible mutual conformations within the patch. We enforce broad coverage of torsion space. We control the size of the sample by using energy criteria to eliminate unlikely configurations, and by clustering similar configurations, resulting in 50 candidates for a patch, a manageable number for docking. Conclusions Using a database of protein dimers for which the bound and unbound structures of the monomers are known, we show that from the unbound patch we are able to generate candidates for docking that approximate the bound structure. In patches where backbone change is small (within 1 Å RMSD of bound), we are able to account for flexibility and generate candidates that are good approximations of the bound state (82% are within 1 Å and 98% are within 1.4 Å RMSD of the bound conformation). We also find that even in cases of moderate backbone flexibility our candidates are able to capture some of the overall shape change. Overall, in 650 of 700 test patches we produce a candidate that is either within 1 Å RMSD of the bound conformation or is closer to the bound state than the unbound is. PMID:21092317

  5. CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area

    PubMed Central

    Terashi, Genki; Takeda-Shitaka, Mayuko

    2015-01-01

    Proteins are flexible, and this flexibility has an essential functional role. Flexibility can be observed in loop regions, rearrangements between secondary structure elements, and conformational changes between entire domains. However, most protein structure alignment methods treat protein structures as rigid bodies. Thus, these methods fail to identify the equivalences of residue pairs in regions with flexibility. In this study, we considered that the evolutionary relationship between proteins corresponds directly to the residue–residue physical contacts rather than the three-dimensional (3D) coordinates of proteins. Thus, we developed a new protein structure alignment method, contact area-based alignment (CAB-align), which uses the residue–residue contact area to identify regions of similarity. The main purpose of CAB-align is to identify homologous relationships at the residue level between related protein structures. The CAB-align procedure comprises two main steps: First, a rigid-body alignment method based on local and global 3D structure superposition is employed to generate a sufficient number of initial alignments. Then, iterative dynamic programming is executed to find the optimal alignment. We evaluated the performance and advantages of CAB-align based on four main points: (1) agreement with the gold standard alignment, (2) alignment quality based on an evolutionary relationship without 3D coordinate superposition, (3) consistency of the multiple alignments, and (4) classification agreement with the gold standard classification. Comparisons of CAB-align with other state-of-the-art protein structure alignment methods (TM-align, FATCAT, and DaliLite) using our benchmark dataset showed that CAB-align performed robustly in obtaining high-quality alignments and generating consistent multiple alignments with high coverage and accuracy rates, and it performed extremely well when discriminating between homologous and nonhomologous pairs of proteins in both

  6. CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area.

    PubMed

    Terashi, Genki; Takeda-Shitaka, Mayuko

    2015-01-01

    Proteins are flexible, and this flexibility has an essential functional role. Flexibility can be observed in loop regions, rearrangements between secondary structure elements, and conformational changes between entire domains. However, most protein structure alignment methods treat protein structures as rigid bodies. Thus, these methods fail to identify the equivalences of residue pairs in regions with flexibility. In this study, we considered that the evolutionary relationship between proteins corresponds directly to the residue-residue physical contacts rather than the three-dimensional (3D) coordinates of proteins. Thus, we developed a new protein structure alignment method, contact area-based alignment (CAB-align), which uses the residue-residue contact area to identify regions of similarity. The main purpose of CAB-align is to identify homologous relationships at the residue level between related protein structures. The CAB-align procedure comprises two main steps: First, a rigid-body alignment method based on local and global 3D structure superposition is employed to generate a sufficient number of initial alignments. Then, iterative dynamic programming is executed to find the optimal alignment. We evaluated the performance and advantages of CAB-align based on four main points: (1) agreement with the gold standard alignment, (2) alignment quality based on an evolutionary relationship without 3D coordinate superposition, (3) consistency of the multiple alignments, and (4) classification agreement with the gold standard classification. Comparisons of CAB-align with other state-of-the-art protein structure alignment methods (TM-align, FATCAT, and DaliLite) using our benchmark dataset showed that CAB-align performed robustly in obtaining high-quality alignments and generating consistent multiple alignments with high coverage and accuracy rates, and it performed extremely well when discriminating between homologous and nonhomologous pairs of proteins in both

  7. Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling.

    PubMed

    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 [Formula: see text]-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 [Formula: see text]/[Formula: see text]-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

  8. 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

  9. Control of stem cell homeostasis via interlocking microRNA and microProtein feedback loops.

    PubMed

    Brandt, Ronny; Xie, Yakun; Musielak, Thomas; Graeff, Moritz; Stierhof, York-Dieter; Huang, Hai; Liu, Chun-Ming; Wenkel, Stephan

    2013-01-01

    Stem cells in the shoot apex of plants produce cells required for the formation of new leaves. Adult leaves are composed of multiple tissue layers arranged along the dorso-ventral (adaxial/abaxial) axis. Class III homeodomain leucine zipper (HD-ZIPIII) transcription factors play an important role in the set-up of leaf polarity in plants. Loss of HD-ZIPIII function results in strongly misshapen leaves and in severe cases fosters the consumption of the apical stem cells, thus causing a growth arrest in mutant plants. HD-ZIPIII mRNA is under tight control by microRNAs 165/166. In addition to the microRNA-action a second layer of regulation is established by LITTLE ZIPPER (ZPR)-type microProteins, which can interact with HD-ZIPIII proteins, forming attenuated protein complexes. Here we show that REVOLUTA (REV, a member of the HD-ZIPIII family) directly regulates the expression of ARGONAUTE10 (AGO10), ZPR1 and ZPR3. Because AGO10 was shown to dampen microRNA165/6 function, REV establishes a positive feedback loop on its own activity. Since ZPR-type microProteins are known to reduce HD-ZIPIII protein activity, REV concomitantly establishes a negative feedback loop. We propose that the interconnection of these microRNA/microProtein feedback loops regulates polarity set-up and stem cell activity in plants.

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

    PubMed Central

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

    2012-01-01

    Summary 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, 1-2 Å, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point > 140 °C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 Å). PMID:22632833

  11. Atomic-Accuracy Prediction of Protein Loop Structures through an RNA-Inspired Ansatz

    PubMed Central

    Das, Rhiju

    2013-01-01

    Consistently predicting biopolymer structure at atomic resolution from sequence alone remains a difficult problem, even for small sub-segments of large proteins. Such loop prediction challenges, which arise frequently in comparative modeling and protein design, can become intractable as loop lengths exceed 10 residues and if surrounding side-chain conformations are erased. Current approaches, such as the protein local optimization protocol or kinematic inversion closure (KIC) Monte Carlo, involve stages that coarse-grain proteins, simplifying modeling but precluding a systematic search of all-atom configurations. This article introduces an alternative modeling strategy based on a ‘stepwise ansatz’, recently developed for RNA modeling, which posits that any realistic all-atom molecular conformation can be built up by residue-by-residue stepwise enumeration. When harnessed to a dynamic-programming-like recursion in the Rosetta framework, the resulting stepwise assembly (SWA) protocol enables enumerative sampling of a 12 residue loop at a significant but achievable cost of thousands of CPU-hours. In a previously established benchmark, SWA recovers crystallographic conformations with sub-Angstrom accuracy for 19 of 20 loops, compared to 14 of 20 by KIC modeling with a comparable expenditure of computational power. Furthermore, SWA gives high accuracy results on an additional set of 15 loops highlighted in the biological literature for their irregularity or unusual length. Successes include cis-Pro touch turns, loops that pass through tunnels of other side-chains, and loops of lengths up to 24 residues. Remaining problem cases are traced to inaccuracies in the Rosetta all-atom energy function. In five additional blind tests, SWA achieves sub-Angstrom accuracy models, including the first such success in a protein/RNA binding interface, the YbxF/kink-turn interaction in the fourth ‘RNA-puzzle’ competition. These results establish all-atom enumeration as an

  12. Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

    PubMed

    Smith, Colin A; Kortemme, Tanja

    2011-01-01

    Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

  13. Substitutions in a flexible loop of horse liver alcohol dehydrogenase hinder the conformational change and unmask hydrogen transfer.

    PubMed

    Ramaswamy, S; Park, D H; Plapp, B V

    1999-10-19

    When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.

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

    SciTech Connect

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

    2009-01-01

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

  15. Sampling multiple scoring functions can improve protein loop structure prediction accuracy.

    PubMed

    Li, Yaohang; Rata, Ionel; Jakobsson, Eric

    2011-07-25

    Accurately predicting loop structures is important for understanding functions of many proteins. In order to obtain loop models with high accuracy, efficiently sampling the loop conformation space to discover reasonable structures is a critical step. In loop conformation sampling, coarse-grain energy (scoring) functions coupling with reduced protein representations are often used to reduce the number of degrees of freedom as well as sampling computational time. However, due to implicitly considering many factors by reduced representations, the coarse-grain scoring functions may have potential insensitivity and inaccuracy, which can mislead the sampling process and consequently ignore important loop conformations. In this paper, we present a new computational sampling approach to obtain reasonable loop backbone models, so-called the Pareto optimal sampling (POS) method. The rationale of the POS method is to sample the function space of multiple, carefully selected scoring functions to discover an ensemble of diversified structures yielding Pareto optimality to all sampled conformations. The POS method can efficiently tolerate insensitivity and inaccuracy in individual scoring functions and thereby lead to significant accuracy improvement in loop structure prediction. We apply the POS method to a set of 4-12-residue loop targets using a function space composed of backbone-only Rosetta and distance-scale finite ideal-gas reference (DFIRE) and a triplet backbone dihedral potential developed in our lab. Our computational results show that in 501 out of 502 targets, the model sets generated by POS contain structure models are within subangstrom resolution. Moreover, the top-ranked models have a root mean square deviation (rmsd) less than 1 A in 96.8, 84.1, and 72.2% of the short (4-6 residues), medium (7-9 residues), and long (10-12 residues) targets, respectively, when the all-atom models are generated by local optimization from the backbone models and are ranked by our

  16. Three dimensional shape comparison of flexible proteins using the local-diameter descriptor

    PubMed Central

    Fang, Yi; Liu, Yu-Shen; Ramani, Karthik

    2009-01-01

    Background Techniques for inferring the functions of the protein by comparing their shape similarity have been receiving a lot of attention. Proteins are functional units and their shape flexibility occupies an essential role in various biological processes. Several shape descriptors have demonstrated the capability of protein shape comparison by treating them as rigid bodies. But this may give rise to an incorrect comparison of flexible protein shapes. Results We introduce an efficient approach for comparing flexible protein shapes by adapting a local diameter (LD) descriptor. The LD descriptor, developed recently to handle skeleton based shape deformations [1], is adapted in this work to capture the invariant properties of shape deformations caused by the motion of the protein backbone. Every sampled point on the protein surface is assigned a value measuring the diameter of the 3D shape in the neighborhood of that point. The LD descriptor is built in the form of a one dimensional histogram from the distribution of the diameter values. The histogram based shape representation reduces the shape comparison problem of the flexible protein to a simple distance calculation between 1D feature vectors. Experimental results indicate how the LD descriptor accurately treats the protein shape deformation. In addition, we use the LD descriptor for protein shape retrieval and compare it to the effectiveness of conventional shape descriptors. A sensitivity-specificity plot shows that the LD descriptor performs much better than the conventional shape descriptors in terms of consistency over a family of proteins and discernibility across families of different proteins. Conclusion Our study provides an effective technique for comparing the shape of flexible proteins. The experimental results demonstrate the insensitivity of the LD descriptor to protein shape deformation. The proposed method will be potentially useful for molecule retrieval with similar shapes and rapid structure

  17. Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach.

    PubMed

    Lu, X; Lu, D; Scully, M F; Kakkar, V V

    2003-06-01

    Integrins are a family of heterodimeric class I transmembrane receptors, many of which bind to the RGD sequence in adhesive proteins and mediate the adhesive interactions of a variety of cells. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with beta1 and beta3 associated integrins and their potency is at least 500-2000 times higher than short RGD peptides. Structural and functional studies suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined flexible loop. The integrin-binding specificity and selectivity of each of the proteins is controlled by amino acid residues in this loop in close vicinity to the RGD-motif. The review includes an overview of the structure and function of snake-venom integrin antagonists. The ability of these proteins to control platelet aggregation, cell adhesion and ligand binding is compared to that of short linear, cyclic RGD-peptides and RGD-containing proteins and the influence of modulation of amino acid residues flanking the RGD motif is also considered. The review is intended to provide insight into the development of novel inhibitors as drugs.

  18. Numerical Analysis of a Flexible Dual Loop Coil and its Experimental Validation for pre-Clinical Magnetic Resonance Imaging of Rodents at 7 T

    NASA Astrophysics Data System (ADS)

    Solis-Najera, S.; Vazquez, F.; Hernandez, R.; Marrufo, O.; Rodriguez, A. O.

    2016-12-01

    A surface radio frequency coil was developed for small animal image acquisition in a pre-clinical magnetic resonance imaging system at 7 T. A flexible coil composed of two circular loops was developed to closely cover the object to be imaged. Electromagnetic numerical simulations were performed to evaluate its performance before the coil construction. An analytical expression of the mutual inductance for the two circular loops as a function of the separation between them was derived and used to validate the simulations. The RF coil is composed of two circular loops with a 5 cm external diameter and was tuned to 300 MHz and 50 Ohms matched. The angle between the loops was varied and the Q factor was obtained from the S11 simulations for each angle. B1 homogeneity was also evaluated using the electromagnetic simulations. The coil prototype was designed and built considering the numerical simulation results. To show the feasibility of the coil and its performance, saline-solution phantom images were acquired. A correlation of the simulations and imaging experimental results was conducted showing a concordance of 0.88 for the B1 field. The best coil performance was obtained at the 90° aperture angle. A more realistic phantom was also built using a formaldehyde-fixed rat phantom for ex vivo imaging experiments. All images showed a good image quality revealing clearly defined anatomical details of an ex vivo rat.

  19. Effective protein model structure refinement by loop modeling and overall relaxation.

    PubMed

    Lee, Gyu Rie; Heo, Lim; Seok, Chaok

    2016-09-01

    Protein structures predicted by state-of-the-art template-based methods may still have errors when the template proteins are not similar enough to the target protein. Overall target structure may deviate from the template structures owing to differences in sequences. Structural information for some local regions such as loops may not be available when there are sequence insertions or deletions. Those structural aspects that originate from deviations from templates can be dealt with by ab initio structure refinement methods to further improve model accuracy. In the CASP11 refinement experiment, we tested three different refinement methods that utilize overall structure relaxation, loop modeling, and quality assessment of multiple initial structures. From this experiment, we conclude that the overall relaxation method can consistently improve model quality. Loop modeling is the most useful when the initial model structure is high quality, with GDT-HA >60. The method that used multiple initial structures further refined the already refined models; the minor improvements with this method raise the issue of problem with the current energy function. Future research directions are also discussed. Proteins 2016; 84(Suppl 1):293-301. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  20. Intracellular segment between transmembrane helices S0 and S1 of BK channel α subunit contains two amphipathic helices connected by a flexible loop

    SciTech Connect

    Shi, Pan; Li, Dong; Lai, Chaohua; Zhang, Longhua; Tian, Changlin

    2013-08-02

    Highlights: •The loop between S0 and S1 of BK channel was overexpressed and purified in DPC. •NMR studies indicated BK-IS1 contained two helices connected by a flexible loop. •Mg{sup 2+} titration of BK-IS1 indicated two possible binding sites of divalent ions. -- Abstract: The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca{sup 2+} and Mg{sup 2+}, as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0–S6) including an extra helix S0. The intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg{sup 2+} coordination. In this study, BK-IS1 (44–113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide {sup 1}H–{sup 15}N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg{sup 2+}. Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.

  1. The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

    SciTech Connect

    Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

    2013-04-22

    Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.

  2. An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets.

    PubMed

    Regev, Ofir; Korman, Maayan; Hecht, Nir; Roth, Ziv; Forer, Nadav; Zarivach, Raz; Gur, Eyal

    2016-10-09

    Pupylation, the bacterial equivalent of ubiquitylation, involves the conjugation of a prokaryotic ubiquitin-like protein (Pup) to protein targets. In contrast to the ubiquitin system, where many ubiquitin ligases exist, a single bacterial ligase, PafA, catalyzes the conjugation of Pup to a wide array of protein targets. As mediators of target recognition by PafA have not been identified, it would appear that PafA alone determines pupylation target selection. Previous studies indicated that broad specificity and promiscuity are indeed inherent PafA characteristics that probably dictate which proteins are selected for degradation by the Pup-proteasome system. Nonetheless, despite the canonical role played by PafA in the Pup-proteasome system, the molecular mechanism that dictates target binding by PafA remains uncharacterized since the discovery of this enzyme about a decade ago. In this study, we report the identification of PafA residues involved in the binding of protein targets. Initially, docking analysis predicted the residues on PafA with high potential for target binding. Mutational and biochemical approaches subsequently confirmed these predictions and identified a series of additional residues located on an extended loop at the edge of the PafA active site. Mutating residues in this loop rendered PafA defective in the pupylation of a wide variety of protein targets but not in its catalytic mechanism, suggesting an important role for this extended loop in the binding of protein targets. As such, these findings pave the way toward an understanding of the molecular determinants that dictate the broad substrate specificity of PafA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Protein flexibility and ligand recognition: challenges for molecular modeling.

    PubMed

    Spyrakis, Francesca; BidonChanal, Axel; Barril, Xavier; Luque, F Javier

    2011-01-01

    The intrinsic dynamics of macromolecules is an essential property to relate the structure of biomolecular systems with their function in the cell. In the field of ligand-receptor recognition, numerous evidences have revealed the limitations of the lock-and-key theory, and the need to elaborate models that take into account the inherent plasticity of biomolecules, such as the induced-fit model or the existence of an ensemble of pre-equilibrated conformations. Depending on the nature of the target system, ligand binding can be associated with small local adjustments in side chains or even the backbone to large-scale motions of structural fragments, domains or even subunits. Reproducing the inherent flexibility of biomolecules has thus become one of the most challenging issues in molecular modeling and simulation studies, as it has direct implications in our understanding of the structure-function relationships, but even in areas such as virtual screening and structure-based drug discovery. Given the intrinsic limitation of conventional simulation tools, only events occurring in short time scales can be reproduced at a high accuracy level through all-atom techniques such as Molecular Dynamics simulations. However, larger structural rearrangements demand the use of enhanced sampling methods relying on modified descriptions of the biomolecular system or the potential surface. This review illustrates the crucial role that structural plasticity plays in mediating ligand recognition through representative examples. In addition, it discusses some of the most powerful computational tools developed to characterize the conformational flexibility in ligand-receptor complexes.

  4. The mechanics of DNA loops bridged by proteins unveiled by single-molecule experiments.

    PubMed

    Tardin, Catherine

    2017-08-10

    Protein-induced DNA bridging and looping is a common mechanism for various and essential processes in bacterial chromosomes. This mechanism is preserved despite the very different bacterial conditions and their expected influence on the thermodynamic and kinetic characteristics of the bridge formation and stability. Over the last two decades, single-molecule techniques carried out on in vitro DNA systems have yielded valuable results which, in combination with theoretical works, have clarified the effects of different parameters of nucleoprotein complexes on the protein-induced DNA bridging and looping process. In this review, I will outline the features that can be measured for such processes with various single-molecule techniques in use in the field. I will then describe both the experimental results and the theoretical models that illuminate the contribution of the DNA molecule itself as well as that of the bridging proteins in the DNA looping mechanism at play in the nucleoid of E. coli. Copyright © 2017. Published by Elsevier B.V.

  5. Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs

    PubMed Central

    2011-01-01

    Background One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Results Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM. Conclusions Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins. PMID:21689388

  6. High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli

    PubMed Central

    Becker, Nicole A.; Maher, L. James

    2015-01-01

    Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells. PMID:26039992

  7. High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli.

    PubMed

    Becker, Nicole A; Maher, L James

    2015-06-09

    Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

  8. Protein-ligand docking with multiple flexible side chains

    NASA Astrophysics Data System (ADS)

    Zhao, Yong; Sanner, Michel F.

    2008-09-01

    In this work, we validate and analyze the results of previously published cross docking experiments and classify failed dockings based on the conformational changes observed in the receptors. We show that a majority of failed experiments (i.e. 25 out of 33, involving four different receptors: cAPK, CDK2, Ricin and HIVp) are due to conformational changes in side chains near the active site. For these cases, we identify the side chains to be made flexible during docking calculation by superimposing receptors and analyzing steric overlap between various ligands and receptor side chains. We demonstrate that allowing these side chains to assume rotameric conformations enables the successful cross docking of 19 complexes (ligand all atom RMSD < 2.0 Å) using our docking software FLIPDock. The number of side receptor side chains interacting with a ligand can vary according to the ligand's size and shape. Hence, when starting from a complex with a particular ligand one might have to extend the region of potential interacting side chains beyond the ones interacting with the known ligand. We discuss distance-based methods for selecting additional side chains in the neighborhood of the known active site. We show that while using the molecular surface to grow the neighborhood is more efficient than Euclidian-distance selection, the number of side chains selected by these methods often remains too large and additional methods for reducing their count are needed. Despite these difficulties, using geometric constraints obtained from the network of bonded and non-bonded interactions to rank residues and allowing the top ranked side chains to be flexible during docking makes 22 out of 25 complexes successful.

  9. Protein flexibility: coordinate uncertainties and interpretation of structural differences

    SciTech Connect

    Rashin, Alexander A.; Rashin, Abraham H. L.; Jernigan, Robert L.

    2009-11-01

    Criteria for the interpretability of coordinate differences and a new method for identifying rigid-body motions and nonrigid deformations in protein conformational changes are developed and applied to functionally induced and crystallization-induced conformational changes. Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functional motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent ‘noise’, showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallographic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function.

  10. Slipknotting upon native-like loop formation in a trefoil knot protein.

    PubMed

    Noel, Jeffrey K; Sułkowska, Joanna I; Onuchic, José N

    2010-08-31

    Protein knots and slipknots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Recent experimental results show that knotting, starting from a fully extended polypeptide, has not yet been observed. Understanding the nucleation process of folding knots is thus a natural challenge for both experimental and theoretical investigation. In this study, we employ energy landscape theory and molecular dynamics to elucidate the entire folding mechanism. The full free energy landscape of a knotted protein is mapped using an all-atom structure-based protein model. Results show that, due to the topological constraint, the protein folds through a three-state mechanism that contains (i) a precise nucleation site that creates a correctly twisted native loop (first barrier) and (ii) a rate-limiting free energy barrier that is traversed by two parallel knot-forming routes. The main route corresponds to a slipknot conformation, a collapsed configuration where the C-terminal helix adopts a hairpin-like configuration while threading, and the minor route to an entropically limited plug motion, where the extended terminus is threaded as through a needle. Knot formation is a late transition state process and results show that random (nonspecific) knots are a very rare and unstable set of configurations both at and below folding temperature. Our study shows that a native-biased landscape is sufficient to fold complex topologies and presents a folding mechanism generalizable to all known knotted protein topologies: knotting via threading a native-like loop in a preordered intermediate.

  11. Id Helix-Loop-Helix Proteins Antagonize Pax Transcription Factor Activity by Inhibiting DNA Binding

    PubMed Central

    Roberts, E. Claire; Deed, Richard W.; Inoue, Toshiaki; Norton, John D.; Sharrocks, Andrew D.

    2001-01-01

    The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family. PMID:11134340

  12. Protein flexibility: coordinate uncertainties and interpretation of structural differences

    PubMed Central

    Rashin, Alexander A.; Rashin, Abraham H. L.; Jernigan, Robert L.

    2009-01-01

    Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functional motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent ‘noise’, showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallo­graphic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function. PMID:19923711

  13. Protein flexibility: coordinate uncertainties and interpretation of structural differences.

    PubMed

    Rashin, Alexander A; Rashin, Abraham H L; Jernigan, Robert L

    2009-11-01

    Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functional motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent 'noise', showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallographic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function.

  14. The effect of elastomer chain flexibility on protein adsorption.

    PubMed

    Vyner, Moira C; Liu, Lina; Sheardown, Heather D; Amsden, Brian G

    2013-12-01

    Cells are known to respond differently when grown on materials of varying stiffness. However, the mechanism by which a cell senses substrate stiffness is unknown. Lower crosslink density elastomers formed from acrylated star-poly(d,l lactide-co-ϵ-caprolactone) have previously been shown to support higher smooth muscle cell proliferation in in vitro culture. This difference in growth was hypothesized to be due to differences in protein adsorption that resulted from differences in polymer chain mobility at the surface. Therefore, layer mass and viscoelastic properties were measured for HSA, IgG, fibronectin, vitronectin, and serum supplemented media adsorbed to elastomers of two crosslink densities. Significantly more fibronectin adsorbed to the lower crosslink density surface while significantly more IgG adsorbed to the higher crosslink density surface. Furthermore, differences in fibronectin and IgG layer shear moduli were observed, suggesting that there was a difference in the conformation of the adsorbed protein. ATR-FTIR analysis showed that the lower crosslink density elastomer absorbed more surface water. The increased amount of water may cause greater entropic gains upon protein adsorption to the lower crosslink density surface, which increases total protein adsorption from serum and may cause differences in protein conformation and thus cell behavior.

  15. Ribosomal Protein S14 Unties the MDM2-p53 Loop Upon Ribosomal Stress

    PubMed Central

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Zhang, Qi; Lu, Hua

    2013-01-01

    The MDM2-p53 feedback loop is crucially important for restricting p53 level and activity during normal cell growth and proliferation, and is thus subjected to dynamic regulation in order for cells to activate p53 upon various stress signals. Several ribosomal proteins, such as RPL11, RPL5, RPL23, RPL26, or RPS7, have been shown to play a role in regulation of this feedback loop in response to ribosomal stress. Here, we identify another ribosomal protein S14, which is highly associated with 5q-syndrome, as a novel activator of p53 by inhibiting MDM2 activity. We found that RPS14, but not RPS19, binds to the central acidic domain of MDM2, like RPL5 and RPL23, and inhibits its E3 ubiquitin ligase activity toward p53. This RPS14-MDM2 binding was induced upon ribosomal stress caused by actinomycin D or mycophenolic acid. Overexpression of RPS14, but not RPS19, elevated p53 level and activity, leading to G1 or G2 arrest. Conversely, knockdown of RPS14 alleviated p53 induction by these two reagents. Interestingly, knockdown of either RPS14 or RPS19 caused a ribosomal stress that led to p53 activation, which was impaired by further knocking down the level of RPL11 or RPL5. Together, our results demonstrate that RPS14 and RPS19 play distinct roles in regulating the MDM2-p53 feedback loop in response to ribosomal stress. PMID:22391559

  16. Anatomy of protein disorder, flexibility and disease-related mutations

    PubMed Central

    Lu, Hui-Chun; Chung, Sun Sook; Fornili, Arianna; Fraternali, Franca

    2015-01-01

    Integration of protein structural information with human genetic variation and pathogenic mutations is essential to understand molecular mechanisms associated with the effects of polymorphisms on protein interactions and cellular processes. We investigate occurrences of non-synonymous SNPs in ordered and disordered protein regions by systematic mapping of common variants and disease-related SNPs onto these regions. We show that common variants accumulate in disordered regions; conversely pathogenic variants are significantly depleted in disordered regions. These different occurrences of pathogenic and common SNPs can be attributed to a negative selection on random mutations in structurally highly constrained regions. New approaches in the study of quantitative effects of pathogenic-related mutations should effectively account for all the possible contexts and relative functional constraints in which the sequence variation occurs. PMID:26322316

  17. Domain Hierarchy and closed Loops (DHcL): a server for exploring hierarchy of protein domain structure

    PubMed Central

    Koczyk, Grzegorz; Berezovsky, Igor N.

    2008-01-01

    Domain hierarchy and closed loops (DHcL) (http://sitron.bccs.uib.no/dhcl/) is a web server that delineates energy hierarchy of protein domain structure and detects domains at different levels of this hierarchy. The server also identifies closed loops and van der Waals locks, which constitute a structural basis for the protein domain hierarchy. The DHcL can be a useful tool for an express analysis of protein structures and their alternative domain decompositions. The user submits a PDB identifier(s) or uploads a 3D protein structure in a PDB format. The results of the analysis are the location of domains at different levels of hierarchy, closed loops, van der Waals locks and their interactive visualization. The server maintains a regularly updated database of domains, closed loop and van der Waals locks for all X-ray structures in PDB. DHcL server is available at: http://sitron.bccs.uib.no/dhcl. PMID:18502776

  18. Protein detection through different platforms of immuno-loop-mediated isothermal amplification

    NASA Astrophysics Data System (ADS)

    Pourhassan-Moghaddam, Mohammad; Rahmati-Yamchi, Mohammad; Akbarzadeh, Abolfazl; Daraee, Hadis; Nejati-Koshki, Kazem; Hanifehpour, Younes; Joo, Sang Woo

    2013-11-01

    Different immunoassay-based methods have been devised to detect protein targets. These methods have some challenges that make them inefficient for assaying ultra-low-amounted proteins. ELISA, iPCR, iRCA, and iNASBA are the common immunoassay-based methods of protein detection, each of which has specific and common technical challenges making it necessary to introduce a novel method in order to avoid their problems for detection of target proteins. Here we propose a new method nominated as `immuno-loop-mediated isothermal amplification' or `iLAMP'. This new method is free from the problems of the previous methods and has significant advantages over them. In this paper we also offer various configurations in order to improve the applicability of this method in real-world sample analyses. Important potential applications of this method are stated as well.

  19. Construction of a linker library with widely controllable flexibility for fusion protein design.

    PubMed

    Li, Gang; Huang, Ziliang; Zhang, Chong; Dong, Bo-Jun; Guo, Ruo-Hai; Yue, Hong-Wei; Yan, Li-Tang; Xing, Xin-Hui

    2016-01-01

    Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.

  20. The BASL Polarity Protein Controls a MAPK Signaling Feedback Loop in Asymmetric Cell Division

    PubMed Central

    Zhang, Ying; Wang, Pengcheng; Shao, Wanchen; Zhu, Jian-Kang; Dong, Juan

    2015-01-01

    SUMMARY Cell polarization is linked to fate determination during asymmetric division of plant stem cells, but the underlying molecular mechanisms remain unknown. In Arabidopsis, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) is polarized to control stomatal asymmetric division. A MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade determines terminal stomatal fate by promoting the degradation of the lineage determinant SPEECHLESS (SPCH). Here we demonstrate that a positive feedback loop between BASL and the MAPK pathway constitutes a polarity module at the cortex. Cortical localization of BASL requires phosphorylation mediated by MPK3/6. Phosphorylated BASL functions as a scaffold and recruits the MAPKKK YODA and MPK3/6 to spatially concentrate signaling at the cortex. Activated MPK3/6 reinforces the feedback loop by phosphorylating BASL, and inhibits stomatal fate by phosphorylating SPCH. Polarization of the BASL-MAPK signaling feedback module represents a mechanism connecting cell polarity to fate differentiation during asymmetric stem cell division in plants. PMID:25843888

  1. Characterizing the existing and potential structural space of proteins by large-scale multiple loop permutations

    PubMed Central

    Dai, Liang; Zhou, Yaoqi

    2011-01-01

    Worldwide structural genomics projects are increasing structure coverage of sequence space but have not significantly expanded the protein structure space itself (i.e. number of unique structural folds) since 2007. Discovering new structural folds experimentally by directed evolution and random recombination of secondary-structure blocks is also proved rarely successful. Meanwhile, previous computational efforts for large-scale mapping of protein structure space are limited to simple model proteins and led to an inconclusive answer on the completeness of the existing, observed protein structure space. Here, we build novel protein structures by extending naturally occurring circular (single-loop) permutation to multiple-loop permutations (MLP). These structures are clustered by structural similarity measure called TM-Score. The computational technique allows us to produce different structural clusters on the same naturally occurring, packed, stable core but with alternatively connected secondary-structure segments. A large-scale MLP of 2936 SCOP domains reproduces those existing structural clusters (63%) mostly as hubs for many non-redundant sequences and illustrates newly discovered novel clusters as islands adopted by a few sequences only. Results further show that there exist a significant number of novel, potentially stable clusters for medium or large-size single-domain proteins, in particular (>100 amino-acid residues) that are either not yet adopted by nature or adopted only by a few sequences. This study suggests that MLP provides a simple yet highly effective tool for engineering and design of novel protein structures (including naturally knotted proteins). The implication of recovering CASP new-fold targets by MLP on template-based structure prediction is also discussed. Our MLP structures are available for download at the publication page of the website http://sparks.informatics.iupui.edu. PMID:21376059

  2. Salvador-Warts-Hippo pathway in a developmental checkpoint monitoring Helix-Loop-Helix proteins

    PubMed Central

    Wang, Lan-Hsin; Baker, Nicholas E.

    2014-01-01

    The E-proteins and Id-proteins are, respectively, the positive and negative heterodimer partners for the basic-helix-loop-helix protein family, and as such contribute to a remarkably large number of cell fate decisions. E-proteins and Id-proteins also function to inhibit or promote cell proliferation and cancer. Using a genetic modifier screen in Drosophila, we show that the Id-protein Extramacrochaetae enables growth by suppressing activation of the Salvador-Warts-Hippo pathway of tumor suppressors, activation that requires transcriptional activation of the expanded gene by the E-protein Daughterless. Daughterless protein binds to an intronic enhancer in the expanded gene, both activating the SWH pathway independently of the transmembrane protein Crumbs, and bypassing the negative feedback regulation that targets the same expanded enhancer. Thus the Salvador-Warts-Hippo pathway has a cell-autonomous function to prevent inappropriate differentiation due to transcription factor imbalance, and monitors the intrinsic developmental status of progenitor cells, distinct from any responses to cell-cell interactions. PMID:25579975

  3. Structural delineation of stem-loop RNA binding by human TAF15 protein

    PubMed Central

    Kashyap, Maruthi; Ganguly, Akshay Kumar; Bhavesh, Neel Sarovar

    2015-01-01

    Human TATA binding protein associated factor 2 N (TAF15) and Fused in sarcoma (FUS) are nucleic acid binding proteins belonging to the conserved FET family of proteins. They are involved in diverse processes such as pre-mRNA splicing, mRNA transport, and DNA binding. The absence of information regarding the structural mechanism employed by the FET family in recognizing and discriminating their cognate and non-cognate RNA targets has hampered the attainment of consensus on modes of protein-RNA binding for this family. Our study provides a molecular basis of this RNA recognition using a combination of solution-state NMR spectroscopy, calorimetry, docking and molecular dynamics simulation. Analysis of TAF15-RRM solution structure and its binding with stem-loop RNA has yielded conclusive evidence of a non-canonical mode of RNA recognition. Rather than classical stacking interactions that occur across nitrogen bases and aromatic amino acids on ribonucleoprotein sites, moderate-affinity hydrogen bonding network between the nitrogen bases in the stem-loop RNA and a concave face on the RRM surface primarily mediate TAF15-RRM RNA interaction. We have compared the binding affinities across a set of single-stranded RNA oligonucleotides to conclusively establish that RNA binding is dependent upon structural elements in the RNA rather than sequence. PMID:26612539

  4. Prediction of flexible/rigid regions from protein sequences using k-spaced amino acid pairs

    PubMed Central

    Chen, Ke; Kurgan, Lukasz A; Ruan, Jishou

    2007-01-01

    Background Traditionally, it is believed that the native structure of a protein corresponds to a global minimum of its free energy. However, with the growing number of known tertiary (3D) protein structures, researchers have discovered that some proteins can alter their structures in response to a change in their surroundings or with the help of other proteins or ligands. Such structural shifts play a crucial role with respect to the protein function. To this end, we propose a machine learning method for the prediction of the flexible/rigid regions of proteins (referred to as FlexRP); the method is based on a novel sequence representation and feature selection. Knowledge of the flexible/rigid regions may provide insights into the protein folding process and the 3D structure prediction. Results The flexible/rigid regions were defined based on a dataset, which includes protein sequences that have multiple experimental structures, and which was previously used to study the structural conservation of proteins. Sequences drawn from this dataset were represented based on feature sets that were proposed in prior research, such as PSI-BLAST profiles, composition vector and binary sequence encoding, and a newly proposed representation based on frequencies of k-spaced amino acid pairs. These representations were processed by feature selection to reduce the dimensionality. Several machine learning methods for the prediction of flexible/rigid regions and two recently proposed methods for the prediction of conformational changes and unstructured regions were compared with the proposed method. The FlexRP method, which applies Logistic Regression and collocation-based representation with 95 features, obtained 79.5% accuracy. The two runner-up methods, which apply the same sequence representation and Support Vector Machines (SVM) and Naïve Bayes classifiers, obtained 79.2% and 78.4% accuracy, respectively. The remaining considered methods are characterized by accuracies below 70

  5. Protein-reactive, thermoresponsive copolymers with high flexibility and biodegradability.

    PubMed

    Guan, Jianjun; Hong, Yi; Ma, Zuwei; Wagner, William R

    2008-04-01

    A family of injectable, biodegradable, and thermosensitive copolymers based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide, and a macromer polylactide-hydroxyethyl methacrylate were synthesized by free radical polymerization. Copolymers were injectable at or below room temperature and formed robust hydrogels at 37 degrees C. The effects of monomer ratio, polylactide length, and AAc content on the chemical and physical properties of the hydrogel were investigated. Copolymers exhibited lower critical solution temperatures (LCSTs) from 18 to 26 degrees C. After complete hydrolysis, hydrogels were soluble in phosphate buffered saline at 37 degrees C with LCSTs above 40.8 degrees C. Incorporation of type I collagen at varying mass fractions by covalent reaction with the copolymer backbone slightly increased LCSTs. Water content was 32-80% without collagen and increased to 230% with collagen at 37 degrees C. Hydrogels were highly flexible and relatively strong at 37 degrees C, with tensile strengths from 0.3 to 1.1 MPa and elongations at break from 344 to 1841% depending on NIPAAm/HEMAPLA ratio, AAc content, and polylactide length. Increasing the collagen content decreased both elongation at break and tensile strength. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Degradation products were shown to be noncytotoxic. Cell adhesion on the hydrogels was 30% of that for tissue culture polystyrene but increased to statistically approximate this control surface after collagen incorporation. These newly described thermoresponsive copolymers demonstrated attractive properties to serve as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications.

  6. A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps.

    PubMed

    Kumar, Mattaparthi Venkata Satish; Swaminathan, Rajaram

    2010-03-01

    The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three-dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

  7. Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences.

    PubMed

    Choudhury, Susobhan; Ghosh, Basusree; Singh, Priya; Ghosh, Raka; Roy, Siddhartha; Pal, Samir Kumar

    2016-07-21

    The nature of the interface of specific protein-DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3-Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DNA.

  8. PcG Proteins, DNA Methylation, and Gene Repression by Chromatin Looping

    PubMed Central

    Tiwari, Vijay K; McGarvey, Kelly M; Licchesi, Julien D.F; Ohm, Joyce E; Herman, James G; Schübeler, Dirk; Baylin, Stephen B

    2008-01-01

    Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream (∼30 kb) of and extending ∼60 kb around one such gene, GATA-4, is organized—in Tera-2 undifferentiated embryonic carcinoma (EC) cells—in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)–mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a ∼60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a

  9. Steric confinement and enhanced local flexibility assist knotting in simple models of protein folding.

    PubMed

    Soler, Miguel A; Rey, Antonio; Faísca, Patrícia F N

    2016-09-29

    The chaperonin complex GroEL-GroES is able to accelerate the folding process of knotted proteins considerably. However, the folding mechanism inside the chaperonin cage is elusive. Here we use a combination of lattice and off-lattice Monte Carlo simulations of simple Gō models to study the effect of physical confinement and local flexibility on the folding process of protein model systems embedding a trefoil knot in their native structure. This study predicts that steric confinement plays a specific role in the folding of knotted proteins by increasing the knotting probability for very high degrees of confinement. This effect is observed for protein MJ0366 even above the melting temperature for confinement sizes compatible with the size of the GroEL/GroES chaperonin cage. An enhanced local flexibility produces the same qualitative effects on the folding process. In particular, we observe that knotting probability increases up to 40% in the transition state of protein MJ0366 when flexibility is enhanced. This is underlined by a structural change in the transition state, which becomes devoid of helical content. No relation between the knotting mechanism and flexibility was found in the context of the off-lattice model adopted in this work.

  10. On the relation between residue flexibility and residue interactions in proteins.

    PubMed

    Yin, Hui; Li, Yi-Zhou; Li, Meng-Long

    2011-05-01

    B-factor from X-ray crystal structure can well measure protein structural flexibility, which plays an important role in different biological processes, such as catalysis, binding and molecular recognition. Understanding the essence of flexibility can be helpful for the further study of the protein function. In this study, we attempted to correlate the flexibility of a residue to its interactions with other residues by representing the protein structure as a residue contact network. Here, several well established network topological parameters were employed to feature such interactions. A prediction model was constructed for B-factor of a residue by using support vector regression (SVR). Pearson correlation coefficient (CC) was used as the performance measure. CC values were 0.63 and 0.62 for single amino acid and for the whole sequence, respectively. Our results revealed well correlations between B-factors and network topological parameters. This suggests that the protein structural flexibility could be well characterized by the inter-amino acid interactions in a protein.

  11. Protein interaction evolution from promiscuity to specificity with reduced flexibility in an increasingly complex network

    PubMed Central

    Alhindi, T.; Zhang, Z.; Ruelens, P.; Coenen, H.; Degroote, H.; Iraci, N.; Geuten, K.

    2017-01-01

    A key question regarding protein evolution is how proteins adapt to the dynamic environment in which they function and how in turn their evolution shapes the protein interaction network. We used extant and resurrected ancestral plant MADS-domain transcription factors to understand how SEPALLATA3, a protein with hub and glue properties, evolved and takes part in network organization. Although the density of dimeric interactions was saturated in the network, many new interactions became mediated by SEPALLATA3 after a whole genome triplication event. By swapping SEPALLATA3 and its ancestors between dimeric networks of different ages, we found that the protein lost the capacity of promiscuous interaction and acquired specificity in evolution. This was accompanied with constraints on conformations through proline residue accumulation, which made the protein less flexible. SHORT VEGETATIVE PHASE on the other hand (non-hub) was able to gain protein-protein interactions due to a C-terminal domain insertion, allowing for a larger interaction interface. These findings illustrate that protein interaction evolution occurs at the level of conformational dynamics, when the binding mechanism concerns an induced fit or conformational selection. Proteins can evolve towards increased specificity with reduced flexibility when the complexity of the protein interaction network requires specificity. PMID:28337996

  12. CD-loop extension in Zika virus envelope protein key for stability and pathogenesis.

    PubMed

    Gallichotte, Emily N; Dinnon, Kenneth H; Lim, Xin-Ni; Ng, Thiam-Seng; Lim, Elisa X Y; Menachery, Vineet D; Lok, Shee-Mei; Baric, Ralph S

    2017-09-08

    With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  13. 3D shape reconstruction of loop objects in X-ray protein crystallography.

    PubMed

    Strutz, Tilo

    2011-01-01

    Knowledge of the shape of crystals can benefit data collection in X-ray crystallography. A preliminary step is the determination of the loop object, i.e., the shape of the loop holding the crystal. Based on the standard set-up of experimental X-ray stations for protein crystallography, the paper reviews a reconstruction method merely requiring 2D object contours and presents a dedicated novel algorithm. Properties of the object surface (e.g., texture) and depth information do not have to be considered. The complexity of the reconstruction task is significantly reduced by slicing the 3D object into parallel 2D cross-sections. The shape of each cross-section is determined using support lines forming polygons. The slicing technique allows the reconstruction of concave surfaces perpendicular to the direction of projection. In spite of the low computational complexity, the reconstruction method is resilient to noisy object projections caused by imperfections in the image-processing system extracting the contours. The algorithm developed here has been successfully applied to the reconstruction of shapes of loop objects in X-ray crystallography.

  14. The role of peptide loops of the Bordetella pertussis protein P.69 pertactin in antibody recognition.

    PubMed

    Hijnen, Marcel; de Voer, Richarda; Mooi, Frits R; Schepp, Rutger; Moret, Ed E; van Gageldonk, Pieter; Smits, Gaby; Berbers, Guy A M

    2007-08-01

    Bordetella pertussis, the etiological agent of whooping cough, is re-emerging in several countries with a traditionally high vaccine uptake. In these B. pertussis strains, polymorphisms were found in several proteins, including P.69 pertactin (P.69 Prn). P.69 Prn, an adhesin, contains two variable regions which are composed of repeats, one of which flanks the receptor binding site. Antibody titers against P.69 Prn correlate with protection and P.69 Prn is one of the components of acellular pertussis vaccines. Nevertheless, little is known about the structure and location of P.69 Prn epitopes. We used a three pronged approach to identify discontinuous epitopes that are recognized by mouse monoclonal antibodies, i.e. site-directed mutagenesis, deletion mapping and competition assays. Site-directed mutagenesis was focused on regions of P.69 Prn predicted to form loops according to the crystal structure. In this report we describe the location of several discontinuous epitopes that are also recognized by human antibodies. Our results reveal an important role of the N-terminus in immune recognition. We provide data for an indirect role of loops in immune evasion by masking of epitopes. We propose that the repeat regions have evolved to allow rapid antigenic variation to deflect the immune response from the functional domain of P.69 Prn. The results presented here provide a better understanding of the structure and function of variable loops and their role in the persistence of pathogens in immunologically primed populations.

  15. Flexibility and rigidity, requirements for the function of proteins and protein pigment complexes. Eleventh Keilin memorial lecture.

    PubMed

    Huber, R

    1987-12-01

    Proteins may be rigid or flexible to various degrees as required for optimum function. Flexibility at the level of amino acid side-chains occurs universally and is important for binding and catalysis. Flexibility of large parts of a protein which rearrange or move are particularly interesting and will be discussed here. We differentiate between certain categories of large-scale flexibility although the boundaries between them are diffuse: flexibility of peptide segments, domain motions and order-disorder transitions of spatially contigous regions. The domains may be flexibly linked to allow rather unrestricted motion or the motion may be constrained to certain modes. The polypeptide segments linking the domains show characteristic structural features. The various categories of flexibility will be illustrated with the following examples. (a) Small protein proteinase inhibitors which are rather rigid molecules which provide binding surfaces complementary to their cognate proteases, but also show limited segmental flexibility and adaptation. (b) Large plasma inhibitors which exhibit large conformational changes upon interaction with proteases probably for regulatory purposes. (c) Pancreatic serine proteases which employ a disorder-order transition of their activation domain as a means to regulate enzymic activity. (d) Immunoglobulins in which rather unrestricted and also hinged domain motions occur in different parts of the molecule probably to allow binding to antigens in different arrangements. (e) Citrate synthase which adopts open and closed forms by a hinged domain motion to bind substrates and release products and to perform the catalytic condensation reaction, respectively. (f) The bifunctional multienzyme complex riboflavin synthase in which two enzymes (alpha and beta) catalyse two consecutive enzymic reactions. The beta-subunits form a shell, in which the alpha-subunits are enclosed. Diffusional motion of the catalytic intermediates is therefore restricted

  16. Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity.

    PubMed Central

    Johnson, J D; Zhang, W; Rudnick, A; Rutter, W J; German, M S

    1997-01-01

    LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues. PMID:9199284

  17. Increasing protein stability by polar surface residues : domain-wide consequences of interactions within a loop.

    SciTech Connect

    Pokkuluri, P. R.; Raffen, R.; Dieckman, L.; Boogaard, C.; Stevens, F. J.; Schiffer, M.; Biosciences Division; C. Boogaard

    2002-01-01

    We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein's function.

  18. Modulation of basic helix-loop-helix transcription complex formation by Id proteins during neuronal differentiation.

    PubMed

    Jögi, Annika; Persson, Paula; Grynfeld, Anna; Påhlman, Sven; Axelson, Håkan

    2002-03-15

    It is assumed that the Id helix-loop-helix (HLH) proteins act by associating with ubiquitously expressed basic HLH (bHLH) transcription factors, such as E47 and E2-2, which prevents these factors from forming functional hetero- or homodimeric DNA binding complexes. Several tissue-specific bHLH proteins, including HASH-1, dHAND, and HES-1, are important for development of the nervous system. Neuroblastoma tumors are derived from the sympathetic nervous system and exhibit neural crest features. In differentiating neuroblastoma cells, HASH-1 is down-regulated, and there is coincident up-regulation of the transcriptional repressor HES-1, which is known to bind the HASH-1 promoter. We found that the three Id proteins expressed in neuroblastoma cells (Id1, Id2, and Id3) were down-regulated during induced differentiation, indicating that Id proteins help keep the tumor cells in an undifferentiated state. Studying interactions, we noted that all four Id proteins could dimerize with E47 or E2-2, but not with HASH-1 or dHAND. However, the Id proteins did complex with HES-1, and increased levels of Id2 reduced the DNA binding activity of HES-1. Furthermore, HES-1 interfered with Id2/E2-2 complex formation. The ability of Id proteins to affect HES-1 activity is of particular interest in neuronal cells, where regulation of HES-1 is essential for the timing of neuronal differentiation.

  19. Flexibility in targeting and insertion during bacterial membrane protein biogenesis

    SciTech Connect

    Bloois, Edwin van; Hagen-Jongman, Corinne M. ten; Luirink, Joen

    2007-10-26

    The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.

  20. In Pursuit of Fully Flexible Protein-Ligand Docking: Modeling the Bilateral Mechanism of Binding.

    PubMed

    Henzler, Angela M; Rarey, Matthias

    2010-03-15

    Modern structure-based drug design aims at accounting for the intrinsic flexibility of therapeutic relevant targets. Over the last few years a considerable amount of docking approaches that encounter this challenging problem has emerged. Here we provide the readership with an overview of established methods for fully flexible protein-ligand docking and current developments in the field. All methods are based on one of two fundamental models which describe the dynamic behavior of proteins upon ligand binding. Methods for ensemble docking (ED) model the protein conformational change before the ligand is placed, whereas induced-fit docking (IFD) optimizes the protein structure afterwards. A third category of docking approaches is formed by recent approaches that follow both concepts. This categorization allows to comprehensively discover strengths and weaknesses of the individual processes and to extract information for their applicability in real world docking scenarios.

  1. Crystal Structures of a Cysteine-modified Mutant in Loop D of Acetylcholine-binding Protein*

    PubMed Central

    Brams, Marijke; Gay, Elaine A.; Sáez, José Colón; Guskov, Albert; van Elk, René; van der Schors, Roel C.; Peigneur, Steve; Tytgat, Jan; Strelkov, Sergei V.; Smit, August B.; Yakel, Jerrel L.; Ulens, Chris

    2011-01-01

    Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET+ and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET+-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET+ produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors. PMID:21115477

  2. Knowledge-Guided Docking of WW Domain Proteins and Flexible Ligands

    NASA Astrophysics Data System (ADS)

    Lu, Haiyun; Li, Hao; Banu Bte Sm Rashid, Shamima; Leow, Wee Kheng; Liou, Yih-Cherng

    Studies of interactions between protein domains and ligands are important in many aspects such as cellular signaling. We present a knowledge-guided approach for docking protein domains and flexible ligands. The approach is applied to the WW domain, a small protein module mediating signaling complexes which have been implicated in diseases such as muscular dystrophy and Liddle’s syndrome. The first stage of the approach employs a substring search for two binding grooves of WW domains and possible binding motifs of peptide ligands based on known features. The second stage aligns the ligand’s peptide backbone to the two binding grooves using a quasi-Newton constrained optimization algorithm. The backbone-aligned ligands produced serve as good starting points to the third stage which uses any flexible docking algorithm to perform the docking. The experimental results demonstrate that the backbone alignment method in the second stage performs better than conventional rigid superposition given two binding constraints. It is also shown that using the backbone-aligned ligands as initial configurations improves the flexible docking in the third stage. The presented approach can also be applied to other protein domains that involve binding of flexible ligand to two or more binding sites.

  3. A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity

    SciTech Connect

    Breustedt, Daniel A.; Chatwell, Lorenz; Skerra, Arne

    2009-10-01

    The crystal structure of tear lipocalin determined in space group P2{sub 1} revealed large structural deviations from the previously solved X-ray structure in space group C2, especially in the loop region and adjoining parts of the β-barrel which give rise to the ligand-binding site. These findings illustrate a novel mechanism for promiscuity in ligand recognition by the lipocalin protein family. Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P2{sub 1} with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 Å resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P2{sub 1} crystal form uncovered major conformational changes (i) in β-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the β-barrel and (iii) in the extended C-terminal segment, which is attached to the β-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family.

  4. Decoupled and linear quadratic regulator control of a large, flexible space antenna with an observer in the control loop

    NASA Technical Reports Server (NTRS)

    Hamer, H. A.; Johnson, K. G.; Young, J. W.

    1985-01-01

    An analysis is performed to compare decoupled and linear quadratic regulator (LQR) procedures for the control of a large, flexible space antenna. Control objectives involve: (1) commanding changes in the rigid-body modes, (2) nulling initial disturbances in the rigid-body modes, or (3) nulling initial disturbances in the first three flexible modes. Control is achieved with two three-axis control-moment gyros located on the antenna column. Results are presented to illustrate various effects on control requirements for the two procedures. These effects include errors in the initial estimates of state variables, variations in the type, number, and location of sensors, and deletions of state-variable estimates for certain flexible modes after control activation. The advantages of incorporating a time lag in the control feedback are also illustrated. In addition, the effects of inoperative-control situations are analyzed with regard to control requirements and resultant modal responses. Comparisons are included which show the effects of perfect state feedback with no residual modes (ideal case). Time-history responses are presented to illustrate the various effects on the control procedures.

  5. Slipknotting upon native-like loop formation in a trefoil knot protein

    PubMed Central

    Noel, Jeffrey K.; Sułkowska, Joanna I.; Onuchic, José N.

    2010-01-01

    Protein knots and slipknots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Recent experimental results show that knotting, starting from a fully extended polypeptide, has not yet been observed. Understanding the nucleation process of folding knots is thus a natural challenge for both experimental and theoretical investigation. In this study, we employ energy landscape theory and molecular dynamics to elucidate the entire folding mechanism. The full free energy landscape of a knotted protein is mapped using an all-atom structure-based protein model. Results show that, due to the topological constraint, the protein folds through a three-state mechanism that contains (i) a precise nucleation site that creates a correctly twisted native loop (first barrier) and (ii) a rate-limiting free energy barrier that is traversed by two parallel knot-forming routes. The main route corresponds to a slipknot conformation, a collapsed configuration where the C-terminal helix adopts a hairpin-like configuration while threading, and the minor route to an entropically limited plug motion, where the extended terminus is threaded as through a needle. Knot formation is a late transition state process and results show that random (nonspecific) knots are a very rare and unstable set of configurations both at and below folding temperature. Our study shows that a native-biased landscape is sufficient to fold complex topologies and presents a folding mechanism generalizable to all known knotted protein topologies: knotting via threading a native-like loop in a preordered intermediate. PMID:20702769

  6. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation.

    PubMed

    Lawrence, Marlon G; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M; Lindahl, Lasse

    2016-07-08

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the ONLY: sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlA(crb) pause peptide. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

    PubMed Central

    Lawrence, Marlon G.; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M.; Lindahl, Lasse

    2016-01-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide. PMID:27257065

  8. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    PubMed

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process.

  9. Intracellular segment between transmembrane helices S0 and S1 of BK channel α subunit contains two amphipathic helices connected by a flexible loop.

    PubMed

    Shi, Pan; Li, Dong; Lai, Chaohua; Zhang, Longhua; Tian, Changlin

    2013-08-02

    The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca(2+) and Mg(2+), as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0-S6) including an extra helix S0. The intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg(2+) coordination. In this study, BK-IS1 (44-113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide (1)H-(15)N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg(2+). Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.

  10. Functions of Replication Protein A as a Sensor of R Loops and a Regulator of RNaseH1.

    PubMed

    Nguyen, Hai Dang; Yadav, Tribhuwan; Giri, Sumanprava; Saez, Borja; Graubert, Timothy A; Zou, Lee

    2017-03-02

    R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.

  11. The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization

    PubMed Central

    He, Jiuya; Mao, Chih-Chieh; Reyes, Aurelio; Sembongi, Hiroshi; Di Re, Miriam; Granycome, Caroline; Clippingdale, Andrew B.; Fearnley, Ian M.; Harbour, Michael; Robinson, Alan J.; Reichelt, Stefanie; Spelbrink, Johannes N.; Walker, John E.; Holt, Ian J.

    2007-01-01

    Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids. PMID:17210950

  12. Suppression of Chondrogenesis by Id Helix-Loop-Helix Proteins in Murine Embryonic Orofacial Tissue

    PubMed Central

    Mukhopadhyay, Partha; Rezzoug, Francine; Webb, Cynthia L.; Pisano, M. Michele; Greene, Robert M.

    2009-01-01

    Inhibitors of differentiation (Id) proteins are helix-loop-helix (HLH) transcription factors lacking a DNA binding domain. Id proteins modulate cell proliferation, apoptosis, and differentiation in embryonic/fetal tissue. Perturbation of any of these processes in cells of the developing orofacial region results in orofacial anomalies. Chondrogenesis, a process integral to normal orofacial ontogenesis, is known to be modulated, in part, by Id proteins. In the present study, the mRNA and protein expression patterns of Id1, Id2, Id3 and Id4 were examined in developing murine orofacial tissue in vivo, as well as in murine embryonic maxillary mesenchymal cells in vitro. The functional role of Ids during chondrogenesis was also explored in vitro. Results reveal that cells derived from developing murine orofacial tissue: (1) express Id1, Id2, Id3 and Id4 mRNAs and proteins on each of gestational days 12-14, (2) express all four Id proteins in a developmentally regulated manner, (3) undergo chondrogenesis and express genes encoding various chondrogenic marker proteins (e.g. Runx2, Type X collagen, Sox9) when cultured under micromass conditions, and (4) can have their chondrogenic potential regulated via alteration of Id protein function through overexpression of a basic HLH factor. In summary, results from the current report reveal for the first time, the expression of all four Id proteins in cells derived from developing murine orofacial tissue, and demonstrate a functional role for the Ids in regulating the ability of these cells to undergo chondrogenesis. PMID:19349107

  13. PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility.

    PubMed

    Zhu, Siying; Henninger, Keely; McGrath, Barbara C; Cavener, Douglas R

    2016-01-01

    PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK's role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility.

  14. Gallus gallus NEU3 sialidase as model to study protein evolution mechanism based on rapid evolving loops

    PubMed Central

    2011-01-01

    Background Large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for protein family evolution. These loops are subjected to lower sequence constraints and can evolve rapidly in novel structural variants. A good model to study this hypothesis is represented by sialidase enzymes. Indeed, the structure of sialidases is a β-propeller composed by anti-parallel β-sheets connected by loops that suit well with the rapid evolving loop hypothesis. These features prompted us to extend our studies on this protein family in birds, to get insights on the evolution of this class of glycohydrolases. Results Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 amino acid sequence mainly constituted by a peptide motif repeated six times in tandem with no homology with any other known protein sequence. The repeat region is located at the same position as the roughly 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved β-strands of the fifth blade of the sialidase β-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence. Conclusion In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved β-sheets and emerging on the opposite site of the catalytic crevice. These data confirm that sialidase family can serve as suitable model for the study of the evolutionary process based on rapid evolving loops, which may had occurred in sialidases. Giving the peculiar organization of the loop region identified in Gg NEU3, this protein can be considered of

  15. Enhanced conformational flexibility of the histone-like (HU) protein from Mycoplasma gallisepticum.

    PubMed

    Altukhov, Dmitry A; Talyzina, Anna A; Agapova, Yulia K; Vlaskina, Anna V; Korzhenevskiy, Dmitry A; Bocharov, Eduard V; Rakitina, Tatiana V; Timofeev, Vladimir I; Popov, Vladimir O

    2016-12-29

    The histone-like (HU) protein is one of the major nucleoid-associated proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This small positively charged dimeric protein binds DNA in a non-sequence specific manner promoting DNA super-structures. The majority of HU proteins are highly conserved among bacteria; however, HU protein from Mycoplasma gallisepticum (HUMgal) has multiple amino acid substitutions in the most conserved regions, which are believed to contribute to its specificity to DNA targets unusual for canonical HU proteins. In this work, we studied the structural dynamic properties of the HUMgal dimer by NMR spectroscopy and MD simulations. The obtained all-atom model displays compliance with the NMR data and confirms the heterogeneous backbone flexibility of HUMgal. We found that HUMgal, being folded into a dimeric conformation typical for HU proteins, has a labile α-helical body with protruded β-stranded arms forming DNA-binding domain that are highly flexible in the absence of DNA. The amino acid substitutions in conserved regions of the protein are likely to affect the conformational lability of the HUMgal dimer that can be responsible for complex functional behavior of HUMgal in vivo, e.g. facilitating its spatial adaptation to non-canonical DNA-targets.

  16. Mutational trends in V3 loop protein sequences observed in different genetic lineages of human immunodeficiency virus type 1.

    PubMed Central

    Korber, B T; MacInnes, K; Smith, R F; Myers, G

    1994-01-01

    Highly variable international human immunodeficiency virus type 1 envelope sequences can be assigned to six major clades, or phylogenetically defined subtypes, designated A through F. These subtypes are approximately equidistant in terms of evolutionary distance measured by nucleotide sequences. This radiation from a common ancestral sequence may have been in step with the spread of the pandemic. In this study, V3 loop protein sequence relationships within these major clades are analyzed to determine how the different lineages might be evolving with respect to this biologically important domain. The V3 loop has been shown to influence viral phenotype and to elicit both humoral and cellular immune responses. To identify patterns in V3 loop amino acid evolution, we cluster the sequences by a phenetic principle which evaluates protein similarities on the basis of amino acid identities and similarities irrespective of evolutionary relationships. When phenetic clustering patterns are superimposed upon phylogenetic subtype classifications, two interesting mutational trends are revealed. First, a set of identical, or highly similar, V3 loop protein sequences can be identified within two otherwise dissimilar genetic subtypes, A and C. Second, the D subtype sequences are found to possess the most radically divergent set of V3 loop sequences. These and other patterns characteristic of the V3 loop reflect the acquisition of specific biological properties during the apparently recent evolution of the human immunodeficiency virus type 1 lineages. PMID:8084005

  17. Coexistence of flexibility and stability of proteins: an equation of state.

    PubMed

    de Leeuw, Marina; Reuveni, Shlomi; Klafter, Joseph; Granek, Rony

    2009-10-09

    We consider a recently suggested "equation of state" for natively folded proteins, and verify its validity for a set of about 5800 proteins. The equation is based on a fractal viewpoint of proteins, on a generalization of the Landau-Peierls instability, and on a marginal stability criterion. The latter allows for coexistence of stability and flexibility of proteins, which is required for their proper function. The equation of state relates the protein fractal dimension d(f), its spectral dimension d(s), and the number of amino acids N. Using structural data from the protein data bank (PDB) and the Gaussian network model (GNM), we compute d(f) and d(s) for the entire set and demonstrate that the equation of state is well obeyed. Addressing the fractal properties and making use of the equation of state may help to engineer biologically inspired catalysts.

  18. Inhibitors bound to Ca(2+)-free sarcoplasmic reticulum Ca(2+)-ATPase lock its transmembrane region but not necessarily its cytosolic region, revealing the flexibility of the loops connecting transmembrane and cytosolic domains.

    PubMed

    Montigny, Cédric; Picard, Martin; Lenoir, Guillaume; Gauron, Carole; Toyoshima, Chikashi; Champeil, Philippe

    2007-12-25

    Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been obtained in the presence of inhibitors such as thapsigargin (TG), bound to the transmembrane region of this protein. Here, we examined the consequences of such binding for the protein. We found that, after TG binding, an active site ligand such as beryllium fluoride can still bind to the ATPase and change the conformation or dynamics of the cytosolic domains (as revealed by the protection afforded against proteolysis), but it becomes unable to induce any change in the transmembrane domain (as revealed by the intrinsic fluorescence of the membranous tryptophan residues). TG also obliterates the Trp fluorescence changes normally induced by binding of MgATP or metal-free ATP, as well as those induced by binding of Mg2+ alone. In the nucleotide binding domain, the environment of Lys515 (as revealed by fluorescein isothiocyanate fluorescence after specific labeling of this residue) is significantly different in the ATPase complex with aluminum fluoride and in the ATPase complex with beryllium fluoride, and in the latter case it is modified by TG. All these facts document the flexibility of the loops connecting the transmembrane and cytosolic domains in the ATPase. In the absence of active site ligands, TG protects the ATPase from cleavage by proteinase K at Thr242-Glu243, suggesting TG-induced reduction in the mobility of these loops. 2,5-Di-tert-butyl-1,4-dihydroxybenzene or cyclopiazonic acid, inhibitors which also bind in or near the transmembrane region, also produce similar overall effects on Ca2+-free ATPase.

  19. tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

    PubMed

    Klassen, Roland; Ciftci, Akif; Funk, Johanna; Bruch, Alexander; Butter, Falk; Schaffrath, Raffael

    2016-12-15

    Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm(5)s(2)U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct(6)A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct(6)A37 or Ψ38/39 and mcm(5)U34 or s(2)U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNA(Lys)UUU or tRNA(Gln)UUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm(5)U34 or s(2)U34, and this defect can be rescued by overexpression of tRNA(Gln)UUG Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Fuzzy domains: new way of describing flexibility and interdependence of the protein domains.

    PubMed

    Yesylevskyy, Semen O; Kharkyanen, Valery N

    2009-03-01

    We proposed the innovative method of domain identification based on the concept of the fuzzy domains. In this method each residue of the protein can belong to several domains simultaneously with certain weights, which reflect to what extent this residue shares the motion pattern of the given domain. Our method allows describing the fuzzy boundaries between the domains and the gradual changes of the motion pattern from one domain to the other. It provides the reasonable compromise between the continuous change of the protein dynamics from one residue to the other and the discrete description of the structure in terms of small number of domains. We suggested quantitative criterion, which shows the overall degree of domain flexibility in the protein. The concept of the fuzzy domains provides an innovative way of visualization of domain flexibility, which makes the gradual transitions between the domains clearly visible and comparable to available experimental and structural data. In the future, the concept of the fuzzy domains can be used in the coarse-grained simulations of the domain dynamics in order to account for internal protein flexibility.

  1. Emerging Behavioral Flexibility in Loop Writing: A longitudinal study in 7- to 9-Year-Old Primary School Children.

    PubMed

    Bosga-Stork, Ida M; Bosga, Jurjen; Meulenbroek, Ruud G J

    2017-04-01

    The development of the ability to adapt one's motor performance to the constraints of a movement task was examined in a longitudinal study involving 7 to-9-year-old children who were asked to perform a preparatory handwriting task. The capacity for sensorimotor synchronization was captured by the standard deviation of the relative phase between pacing signals and writing movements and the capacity to adjust wrist-finger coordination while performing repetitive movements was analyzed by autocorrelations of the vertical pen-tip displacements. While the capacity for synchronization improved with age, the autocorrelations were positive at short time lags only and hardly changed with age. A measure of "the long-term memory" of time series (Hurst exponent) confirmed that the findings were systematic rather than noise. Collectively, the results indicate that flexible movement strategies emerge early on in the first 3 years of formal handwriting education. Implications for educational and clinical practice are considered.

  2. A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

    PubMed

    Zgiby, S; Plater, A R; Bates, M A; Thomson, G J; Berry, A

    2002-01-11

    Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value

  3. Optimum length and flexibility of reovirus attachment protein σ1 are required for efficient viral infection.

    PubMed

    Bokiej, Magdalena; Ogden, Kristen M; Ikizler, Mine; Reiter, Dirk M; Stehle, Thilo; Dermody, Terence S

    2012-10-01

    Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.

  4. Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin.

    PubMed

    Ito, Kohji; Yamaguchi, Yukie; Yanase, Kenji; Ichikawa, Yousuke; Yamamoto, Keiichi

    2009-12-22

    Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected V(max) and K(app) of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K(app) and the velocity, although it did not affect V(max). Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.

  5. Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy.

    PubMed

    Barthelmes, Dominic; Gränz, Markus; Barthelmes, Katja; Allen, Karen N; Imperiali, Barbara; Prisner, Thomas; Schwalbe, Harald

    2015-11-01

    We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured (1)H, (15)N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm(3+)-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1β-S2R2 and the respective single-loop-LBT constructs IL1β-S2, IL1β-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd(3+) as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

  6. The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop

    PubMed Central

    1996-01-01

    No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it. PMID:8609161

  7. DNA replication catalyzed by herpes simplex virus type 1 proteins reveals trombone loops at the fork.

    PubMed

    Bermek, Oya; Willcox, Smaranda; Griffith, Jack D

    2015-01-30

    Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.

  8. Evaluation of the novel algorithm of flexible ligand docking with moveable target-protein atoms.

    PubMed

    Sulimov, Alexey V; Zheltkov, Dmitry A; Oferkin, Igor V; Kutov, Danil C; Katkova, Ekaterina V; Tyrtyshnikov, Eugene E; Sulimov, Vladimir B

    2017-01-01

    We present the novel docking algorithm based on the Tensor Train decomposition and the TT-Cross global optimization. The algorithm is applied to the docking problem with flexible ligand and moveable protein atoms. The energy of the protein-ligand complex is calculated in the frame of the MMFF94 force field in vacuum. The grid of precalculated energy potentials of probe ligand atoms in the field of the target protein atoms is not used. The energy of the protein-ligand complex for any given configuration is computed directly with the MMFF94 force field without any fitting parameters. The conformation space of the system coordinates is formed by translations and rotations of the ligand as a whole, by the ligand torsions and also by Cartesian coordinates of the selected target protein atoms. Mobility of protein and ligand atoms is taken into account in the docking process simultaneously and equally. The algorithm is realized in the novel parallel docking SOL-P program and results of its performance for a set of 30 protein-ligand complexes are presented. Dependence of the docking positioning accuracy is investigated as a function of parameters of the docking algorithm and the number of protein moveable atoms. It is shown that mobility of the protein atoms improves docking positioning accuracy. The SOL-P program is able to perform docking of a flexible ligand into the active site of the target protein with several dozens of protein moveable atoms: the native crystallized ligand pose is correctly found as the global energy minimum in the search space with 157 dimensions using 4700 CPU ∗ h at the Lomonosov supercomputer.

  9. Introducing a rigid loop structure from deer into mouse prion protein increases its propensity for misfolding in vitro.

    PubMed

    Kyle, Leah M; John, Theodore R; Schätzl, Hermann M; Lewis, Randolph V

    2013-01-01

    Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrP(c)) into the disease-associated isoform (PrP(Sc)) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids.

  10. Protein conformational changes studied by diffusion NMR spectroscopy: Application to helix-loop-helix calcium binding proteins

    PubMed Central

    Weljie, Aalim M.; Yamniuk, Aaron P.; Yoshino, Hidenori; Izumi, Yoshinobu; Vogel, Hans J.

    2003-01-01

    Pulsed-field gradient (PFG) diffusion NMR spectroscopy studies were conducted with several helix-loop-helix regulatory Ca2+-binding proteins to characterize the conformational changes associated with Ca2+-saturation and/or binding targets. The calmodulin (CaM) system was used as a basis for evaluation, with similar hydrodynamic radii (Rh) obtained for apo- and Ca2+-CaM, consistent with previously reported Rh data. In addition, conformational changes associated with CaM binding to target peptides from myosin light chain kinase (MLCK), phosphodiesterase (PDE), and simian immunodeficiency virus (SIV) were accurately determined compared with small-angle X-ray scattering results. Both sets of data demonstrate the well-established collapse of the extended Ca2+-CaM molecule into a globular complex upon peptide binding. The Rh of CaM complexes with target peptides from CaM-dependent protein kinase I (CaMKI) and an N-terminal portion of the SIV peptide (SIV-N), as well as the anticancer drug cisplatin were also determined. The CaMKI complex demonstrates a collapse analogous to that observed for MLCK, PDE, and SIV, while the SIV-N shows only a partial collapse. Interestingly, the covalent CaM–cisplatin complex shows a near complete collapse, not expected from previous studies. The method was extended to related calcium binding proteins to show that the Rh of calcium and integrin binding protein (CIB), calbrain, and the calcium-binding region from soybean calcium-dependent protein kinase (CDPK) decrease on Ca2+-binding to various extents. Heteronuclear NMR spectroscopy suggests that for CIB and calbrain this is likely because of shifting the equilibrium from unfolded to folded conformations, with calbrain forming a dimer structure. These results demonstrate the utility of PFG-diffusion NMR to rapidly and accurately screen for molecular size changes on protein–ligand and protein–protein interactions for this class of proteins. PMID:12538886

  11. Flexible programming of cell-free protein synthesis using magnetic bead-immobilized plasmids.

    PubMed

    Lee, Ka-Young; Lee, Kyung-Ho; Park, Ji-Woong; Kim, Dong-Myung

    2012-01-01

    The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis.

  12. Monte Carlo simulations of flexible polyanions complexing with whey proteins at their isoelectric point

    NASA Astrophysics Data System (ADS)

    de Vries, R.

    2004-02-01

    Electrostatic complexation of flexible polyanions with the whey proteins α-lactalbumin and β-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Hückel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that α-lactalbumin complexes much more strongly than β-lactoglobulin. For α-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for β-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches.

  13. Structural mechanism of nuclear transport mediated by importin β and flexible amphiphilic proteins.

    PubMed

    Yoshimura, Shige H; Kumeta, Masahiro; Takeyasu, Kunio

    2014-12-02

    Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of various proteins through the nuclear pore complex (NPC), although they are substantially larger than the size limit of the NPC.To elucidate the molecular mechanism underlying this paradoxical function, we focused on the unique structures called HEAT repeats, which consist of repetitive amphiphilic α helices. An in vitro transport assay and FRAP analyses demonstrated that not only karyopherin β family proteins but also other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport mediators for their binding partners. Biochemical and spectroscopic analyses and molecular dynamics simulations of purified HEAT-rich proteins revealed that they interact with hydrophobic groups, including phenyl and alkyl groups, and undergo reversible conformational changes in tertiary structures, but not in secondary structures. These results show that conformational changes in the flexible amphiphilic motifs play a critical role in translocation through the NPC.

  14. The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein.

    PubMed

    Sonati, Tiziana; Reimann, Regina R; Falsig, Jeppe; Baral, Pravas Kumar; O'Connor, Tracy; Hornemann, Simone; Yaganoglu, Sine; Li, Bei; Herrmann, Uli S; Wieland, Barbara; Swayampakula, Mridula; Rahman, Muhammad Hafizur; Das, Dipankar; Kav, Nat; Riek, Roland; Liberski, Pawel P; James, Michael N G; Aguzzi, Adriano

    2013-09-05

    Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably

  15. Structure-based ligand design for flexible proteins: Application of new F-DycoBlock

    NASA Astrophysics Data System (ADS)

    Zhu, Jiang; Fan, Hao; Liu, Haiyan; Shi, Yunyu

    2001-11-01

    A method of structure-based ligand design - DycoBlock - has been proposed and tested by Liu et al.[1]. It was further improved by Zhu et al. and applied to design new selective inhibitors of cyclooxygenase 2 [2]. In the current work, we present a new methodology - F-DycoBlock that allows for the incorporation of receptor flexibility. During the designing procedure, both the receptor and molecular building blocks are subjected to the multiple-copy stochastic molecular dynamics (MCSMD) simulation [1], while the protein moves in the mean field of all copies. It is tested for two enzymes studied previously - cyclooxygenase 2 (COX-2) and human immunodeficiency type 1 (HIV-1) protease. To identify the applicability of F-DycoBlock, the binding protein structure was used as starting point to explore the conformational space around the bound state. This method can be easily extended to accommodate the flexibility in different degree. Four types of treatment of the receptor flexibility - all-atom restrained, backbone restrained, intramolecular hydrogen-bond restrained and active-site flexible - were tested with or without the grid approximation. Two inhibitors, SC-558 for COX-2 and L700417 for HIV-1 protease, are used in this testing study for comparison with previous results. The accuracy of recovery, binding energy, solvent accessible surface area (SASA) and positional root-mean-square (RMS) deviation are used as criteria. The results indicate that F-DycoBlock is a robust methodology for flexible drug design. It is particularly notable that the protein flexibility has been perfectly associated with each stage of drug design - search for the binding sites, dynamic assembly and optimization of candidate compounds. When all protein atoms were restrained, F-DycoBlock yielded higher accuracy of recovery than DycoBlock (100%). If backbone atoms were restrained, the same ratio of accuracy was achieved. Moreover, with the intramolecular hydrogen bonds restrained, reasonable

  16. Impact of protein binding cavity volume (PCV) and ligand volume (LV) in rigid and flexible docking of protein-ligand complexes.

    PubMed

    Saranya, N; Jeyakanthan, J; Selvaraj, S

    2012-12-15

    The importance of protein binding cavity volume (PCV) and ligand volume (LV) in rigid and flexible docking has been studied in 48 protein-ligand complexes belonging to eight protein families. In continuation of our earlier study on protein flexibility in relationship to PCV and LV, this study analyzes the importance of PCV and LV in the scoring and ranking of ligands in docking experiments. Crystal structures of protein-ligand complexes with varied PCV were chosen for docking ligands of varied volume in each protein family. Docking and scoring accuracy have been evaluated by self and cross docking of ligands to the given protein conformation. Effect of PCV and LV in rigid and flexible docking has been studied both in apo and holo proteins. Rigid docking has performed well when appropriate protein conformation is used. Selecting the proteins with appropriate PCV based on the LV information is suggested for better results in ensemble docking.

  17. Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication

    PubMed Central

    Tucker, Lynne D.; Asara, John M.; Cheruiyot, Collins K.; Lu, Huafei; Wu, Zhijin J.; Newstein, Michael C.; Dooner, Mark S.; Friedman, Jennifer; Lally, Michelle A.; Ramratnam, Bharat

    2016-01-01

    A rare subset of HIV-1–infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1–infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1–infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection. PMID:27454292

  18. Insights into protein flexibility: The relationship between normal modes and conformational change upon protein–protein docking

    PubMed Central

    Dobbins, Sara E.; Lesk, Victor I.; Sternberg, Michael J. E.

    2008-01-01

    Understanding protein interactions has broad implications for the mechanism of recognition, protein design, and assigning putative functions to uncharacterized proteins. Studying protein flexibility is a key component in the challenge of describing protein interactions. In this work, we characterize the observed conformational change for a set of 20 proteins that undergo large conformational change upon association (>2 Å Cα RMSD) and ask what features of the motion are successfully reproduced by the normal modes of the system. We demonstrate that normal modes can be used to identify mobile regions and, in some proteins, to reproduce the direction of conformational change. In 35% of the proteins studied, a single low-frequency normal mode was found that describes well the direction of the observed conformational change. Finally, we find that for a set of 134 proteins from a docking benchmark that the characteristic frequencies of normal modes can be used to predict reliably the extent of observed conformational change. We discuss the implications of the results for the mechanics of protein recognition. PMID:18641126

  19. Gene repression by minimal lac loops in vivo.

    PubMed

    Bond, Laura M; Peters, Justin P; Becker, Nicole A; Kahn, Jason D; Maher, L James

    2010-12-01

    The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.

  20. SA-Mot: a web server for the identification of motifs of interest extracted from protein loops.

    PubMed

    Regad, Leslie; Saladin, Adrien; Maupetit, Julien; Geneix, Colette; Camproux, Anne-Claude

    2011-07-01

    The detection of functional motifs is an important step for the determination of protein functions. We present here a new web server SA-Mot (Structural Alphabet Motif) for the extraction and location of structural motifs of interest from protein loops. Contrary to other methods, SA-Mot does not focus only on functional motifs, but it extracts recurrent and conserved structural motifs involved in structural redundancy of loops. SA-Mot uses the structural word notion to extract all structural motifs from uni-dimensional sequences corresponding to loop structures. Then, SA-Mot provides a description of these structural motifs using statistics computed in the loop data set and in SCOP superfamily, sequence and structural parameters. SA-Mot results correspond to an interactive table listing all structural motifs extracted from a target structure and their associated descriptors. Using this information, the users can easily locate loop regions that are important for the protein folding and function. The SA-Mot web server is available at http://sa-mot.mti.univ-paris-diderot.fr.

  1. Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility

    PubMed Central

    Tomatis, Pablo E.; Fabiane, Stella M.; Simona, Fabio; Carloni, Paolo; Sutton, Brian J.; Vila, Alejandro J.

    2008-01-01

    Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by β-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-β-lactamase (MβL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MβLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective. PMID:19098096

  2. Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility.

    PubMed

    Tomatis, Pablo E; Fabiane, Stella M; Simona, Fabio; Carloni, Paolo; Sutton, Brian J; Vila, Alejandro J

    2008-12-30

    Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by beta-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-beta-lactamase (MbetaL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MbetaLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.

  3. Structure and dynamics of [gamma]-SNAP: Insight into flexibility of proteins from the SNAP family

    SciTech Connect

    Bitto, Eduard; Bingman, Craig A.; Kondrashov, Dmitry A.; McCoy, Jason G.; Bannen, Ryan M.; Wesenberg, Gary E.; Phillips, Jr., George N.

    2010-02-19

    Soluble N-ethylmaleimide-sensitive factor attachment protein gamma ({gamma}-SNAP) is a member of an eukaryotic protein family involved in intracellular membrane trafficking. The X-ray structure of Brachydanio rerio {gamma}-SNAP was determined to 2.6 {angstrom} and revealed an all-helical protein comprised of an extended twisted-sheet of helical hairpins with a helical-bundle domain on its carboxy-terminal end. Structural and conformational differences between multiple observed {gamma}-SNAP molecules and Sec17, a SNAP family protein from yeast, are analyzed. Conformational variation in {gamma}-SNAP molecules is matched with great precision by the two lowest frequency normal modes of the structure. Comparison of the lowest-frequency modes from {gamma}-SNAP and Sec17 indicated that the structures share preferred directions of flexibility, corresponding to bending and twisting of the twisted sheet motif. We discuss possible consequences related to the flexibility of the SNAP proteins for the mechanism of the 20S complex disassembly during the SNAP receptors recycling.

  4. Tryptophan phosphorescence as a monitor of flexibility of membrane proteins in cells

    NASA Astrophysics Data System (ADS)

    Mazhul, Vladimir M.; Scherbin, Dmitry G.

    1997-05-01

    Method of room temperature tryptophan phosphorescence (RTTP) has been used to study slow intramolecular equilibrium motions in membrane proteins. The conventional home-made instruments were employed for measurement of RTTP kinetic and spectral parameters. Objects of the investigation were suspensions of human erythrocyte membranes, different animal and plant cells. On rat gepathocytes it has been shown that membrane proteins in composition of subcellular structures and native cells are able to the RTTP with tens and hundreds milliseconds lifetimes. An overwhelming part of soluble proteins of cytoplasm, karyoplasm and mitochondrial matrix has not capability to RTTP with lifetimes above 1 ms. It is concluded that unlike membrane proteins soluble proteins as a rule are characterized by motions of protein structure with intensive low frequency and large amplitude, that leads to pronounced quenching of their RTTP. In the case of membrane proteins, which are capable of phosphorescence in a millisecond range, the flexibility of the chromophores environment decreases. These results indicate that RTTP method gives the unique possibility to investigate dynamical structure of membrane proteins without their preliminary isolation from cells. The data on membrane proteins intramolecular dynamics in composition of cells at the action of biological active substances in physiological concentrations--Concavalin A, nerve growth factor, epidermal growth factor, 24-epibrassinosteroid received by the phosphorescent method are presented.

  5. The folding state of the lumenal loop determines the thermal stability of light-harvesting chlorophyll a/b protein.

    PubMed

    Mick, Vera; Geister, Sonja; Paulsen, Harald

    2004-11-23

    The major light-harvesting protein of photosystem II (LHCIIb) is the most abundant chlorophyll-binding protein in the thylakoid membrane. It contains three membrane-spanning alpha helices; the first and third one closely interact with each other to form a super helix, and all three helices bind most of the pigment cofactors. The protein loop domains connecting the alpha helices also play an important role in stabilizing the LHCIIb structure. Single amino acid exchanges in either loop were found to be sufficient to significantly destabilize the complex assembled in vitro [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093. Mick, V., Eggert, K., Heinemann, B., Geister, S., and Paulsen, H (2004) Biochemistry 43, 5467-5473]. This work presents an analysis of such point mutations in the lumenal loop with regard to the extent and nature of their effect on LHCIIb stability to obtain detailed information on the contribution of this loop to stabilizing the complex. Most of the mutant proteins yielded pigment-protein complexes if their reconstitution and/or isolation was performed under mild conditions; however, the yields were significantly different. Several mutations in the vicinity of W97 in the N-proximal section of the loop gave low reconstitution yields even under very mild conditions. This confirms our earlier notion that W97 may be of particular relevance in stabilizing LHCIIb. The same amino acid exchanges accelerated thermal complex dissociation in the absence of lithium dodecyl sulfate (LDS) and raised the accessibility of the lumenal loop to protease; both effects were well correlated with the reduction in reconstitution yields. We conclude that a detachment of the lumenal loop is a possible first step in the dissociation of LHCIIb. Dramatically reduced complex yields in the presence but not in the absence of LDS were observed for some but not all mutants, particularly those near the C-proximal end of the loop. We conclude that complex

  6. How different are structurally flexible and rigid binding sites? Sequence and structural features discriminating proteins that do and do not undergo conformational change upon ligand binding.

    PubMed

    Gunasekaran, Kannan; Nussinov, Ruth

    2007-01-05

    Proteins are dynamic molecules and often undergo conformational change upon ligand binding. It is widely accepted that flexible loop regions have a critical functional role in enzymes. Lack of consideration of binding site flexibility has led to failures in predicting protein functions and in successfully docking ligands with protein receptors. Here we address the question: which sequence and structural features distinguish the structurally flexible and rigid binding sites? We analyze high-resolution crystal structures of ligand bound (holo) and free (apo) forms of 41 proteins where no conformational change takes place upon ligand binding, 35 examples with moderate conformational change, and 22 cases where a large conformational change has been observed. We find that the number of residue-residue contacts observed per-residue (contact density) does not distinguish flexible and rigid binding sites, suggesting a role for specific interactions and amino acids in modulating the conformational changes. Examination of hydrogen bonding and hydrophobic interactions reveals that cases that do not undergo conformational change have high polar interactions constituting the binding pockets. Intriguingly, the large, aromatic amino acid tryptophan has a high propensity to occur at the binding sites of examples where a large conformational change has been noted. Further, in large conformational change examples, hydrophobic-hydrophobic, aromatic-aromatic, and hydrophobic-polar residue pair interactions are dominant. Further analysis of the Ramachandran dihedral angles (phi, psi) reveals that the residues adopting disallowed conformations are found in both rigid and flexible cases. More importantly, the binding site residues adopting disallowed conformations clustered narrowly into two specific regions of the L-Ala Ramachandran map. Examination of the dihedral angles changes upon ligand binding shows that the magnitude of phi, psi changes are in general minimal, although some large

  7. Docking ligands into flexible and solvated macromolecules. 7. Impact of protein flexibility and water molecules on docking-based virtual screening accuracy.

    PubMed

    Therrien, Eric; Weill, Nathanael; Tomberg, Anna; Corbeil, Christopher R; Lee, Devin; Moitessier, Nicolas

    2014-11-24

    The use of predictive computational methods in the drug discovery process is in a state of continual growth. Over the last two decades, an increasingly large number of docking tools have been developed to identify hits or optimize lead molecules through in-silico screening of chemical libraries to proteins. In recent years, the focus has been on implementing protein flexibility and water molecules. Our efforts led to the development of Fitted first reported in 2007 and further developed since then. In this study, we wished to evaluate the impact of protein flexibility and occurrence of water molecules on the accuracy of the Fitted docking program to discriminate active compounds from inactive compounds in virtual screening (VS) campaigns. For this purpose, a total of 171 proteins cocrystallized with small molecules representing 40 unique enzymes and receptors as well as sets of known ligands and decoys were selected from the Protein Data Bank (PDB) and the Directory of Useful Decoys (DUD), respectively. This study revealed that implementing displaceable crystallographic or computationally placed particle water molecules and protein flexibility can improve the enrichment in active compounds. In addition, an informed decision based on library diversity or research objectives (hit discovery vs lead optimization) on which implementation to use may lead to significant improvements.

  8. Multiscale Monte Carlo Sampling of Protein Sidechains: Application to Binding Pocket Flexibility

    PubMed Central

    Nilmeier, Jerome; Jacobson, Matt

    2008-01-01

    We present a Monte Carlo sidechain sampling procedure and apply it to assessing the flexibility of protein binding pockets. We implemented a multiple “time step” Monte Carlo algorithm to optimize sidechain sampling with a surface generalized Born implicit solvent model. In this approach, certain forces (those due to long-range electrostatics and the implicit solvent model) are updated infrequently, in “outer steps”, while short-range forces (covalent, local nonbonded interactions) are updated at every “inner step”. Two multistep protocols were studied. The first protocol rigorously obeys detailed balance, and the second protocol introduces an approximation to the solvation term that increases the acceptance ratio. The first protocol gives a 10-fold improvement over a protocol that does not use multiple time steps, while the second protocol generates comparable ensembles and gives a 15-fold improvement. A range of 50–200 inner steps per outer step was found to give optimal performance for both protocols. The resultant method is a practical means to assess sidechain flexibility in ligand binding pockets, as we illustrate with proof-of-principle calculations on six proteins: DB3 antibody, thermolysin, estrogen receptor, PPAR-γ, PI3 kinase, and CDK2. The resulting sidechain ensembles of the apo binding sites correlate well with known induced fit conformational changes and provide insights into binding pocket flexibility. PMID:19119325

  9. A relational database of protein structures designed for flexible enquiries about conformation.

    PubMed

    Islam, S A; Sternberg, M J

    1989-03-01

    A relational database of protein structure has been developed to enable rapid and flexible enquiries about the occurrence of many aspects of protein architecture. The coordinates of 294 proteins from the Brookhaven Data Bank have been processed by standard computer programs to generate many additional terms that quantify aspects of protein structure. These terms include solvent accessibility, main-chain and side-chain dihedral angles, and secondary structure. In a relational database, the information is stored in tables with columns holding the different terms and rows holding the different entries for the terms. The different relational base tables store the information about the protein coordinate set, the different chains in the protein, the amino acid residues and ligands, the atomic coordinates, the salt bridges, the hydrogen bonds, the disulphide bridges and the close tertiary contacts. The database was established under ORACLE management system. Enquiries are constructed in ORACLE using SQL (structured query language) which is simple to use and alleviates the need for extensive computer programs. A single table can be searched for entries that meet various criteria, e.g. all protein solved to better than a given resolution. The power of the database occurs when several tables, or the entries in a single table, are cross-correlated. For example the dihedral angles of proline in the fourth position in an alpha-helix in high resolution structures can be rapidly obtained. The structural database provides a powerful tool to obtain empirical rules about protein conformation. This database of protein structures is part of a joint project between Birkbeck College and Leeds University to establish an integrated data resource of protein sequences and structures (ISIS) that encodes the complex patterns of residues and coordinates that define protein conformation. The entire data resource (ISIS) will provide a system to guide all areas of protein modelling including

  10. Myc/Max and other helix-loop-helix/leucine zipper proteins bend DNA toward the minor groove.

    PubMed Central

    Fisher, D E; Parent, L A; Sharp, P A

    1992-01-01

    A distinct family of DNA-binding proteins is characterized by the presence of adjacent "basic," helix-loop-helix, and leucine zipper domains. Members of this family include the Myc oncoproteins, their binding partner Max, and the mammalian transcription factors USF, TFE3, and TFEB. Consistent with their homologous domains, these proteins bind to DNA containing the same core hexanucleotide sequence CACGTG. Analysis of the conformation of DNA in protein-DNA complexes has been undertaken with a circular permutation assay. Large mobility anomalies were detected for all basic/helix-loop-helix/leucine zipper proteins tested, suggesting that each protein induced a similar degree of bending. Phasing analysis revealed that basic/helix-loop-helix/leucine zipper proteins orient the DNA bend toward the minor groove. The presence of in-phase spacing between adjacent binding sites for this family of proteins in the immunoglobulin heavy-chain enhancer suggests the possible formation of an unusual triple-bended structure and may have implications for the activities of Myc. Images PMID:1465398

  11. Improving protein crystal quality by selective removal of a Ca{sup 2+}-dependent membrane-insertion loop

    SciTech Connect

    Neau, David B.; Gilbert, Nathaniel C.; Bartlett, Sue G.; Dassey, Adam; Newcomer, Marcia E.

    2007-11-01

    Protein engineering dramatically enhances the quality of crystals of a Ca{sup 2+}-dependent membrane-binding protein. Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca{sup 2+}-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 Å crystal structure of 8R-LOX and spectroscopic data suggested that Ca{sup 2+} stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Δ41–45:GSLOX) was engineered. Removal of the membrane-insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 Å improvement in the resolution of the diffraction data.

  12. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1

    SciTech Connect

    Desprez, P.; Hara, E.; Bissell, M.J.

    1995-06-01

    Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses {beta}-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). The authors show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. The authors suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. 53 refs., 6 figs.

  13. The extracellular loop of IRT1 ZIP protein--the chosen one for zinc?

    PubMed

    Potocki, Slawomir; Valensin, Daniela; Camponeschi, Francesca; Kozlowski, Henryk

    2013-10-01

    Zinc complexes with the extracellular loop of IRT1 (iron-regulated transporter 1), a ZIP (ZRT/IRT - Related Protein) family protein from Arabidopsis thaliana, have been studied. This unstructured fragment is responsible for metal selectivity and is located between the II and III transmembrane domains of IRT1. Zinc complexes with the Ac-(95)MHVLPDSFEMLSSICLEENPWHK(117)-NH2 peptide (IRT1), revealed surprisingly high thermodynamic stability. Additionally, an N-terminal fragment of human/mouse ZIP 13 zinc transporter (MPGCPCPGCGMACPR-NH2, later called ZIP13+C), has been chosen for the thermodynamic stability comparison studies. The relative ZIP13+C stability has been shown using several Zn(2+) complexes with artificially arranged multi-cysteine sequences. An interesting coordination mode has been proposed for the IRT1-Zn(2+) complex, in which imidazoles from two histidines (His-96 and His-116), a cysteine thiolate (Cys-109) and one of a glutamic acid carboxyl group are involved. All data were collected using potentiometric, NMR and mass spectrometric methods.

  14. Analyses of inter- and intra-patient variation in the V3 loop of the HIV-1 envelope protein

    SciTech Connect

    Korber, B.; Myers, G.; Wolinsky, S.

    1991-09-17

    The third hypervariable domain of the HIV-1 gp120 envelope protein (V3) has been the focus of intensive sequencing efforts. To date, nearly one thousand V3 loop sequences have been stored in the HIV sequence database. Studies have revealed that the V3 loop elicits potent type-specific immune responses, and that it plays a significant role in cell tropism and fusion . The immunogenic tip of the loop can serve as a type-specific neutralizing antibody epitope, as well as a cytotoxic T-cell epitope. A helper T-cell epitope that lies within the amino terminal half of the V3 loop has also been characterized. Despite the richness of the immunologic response to this region, its potential for variation makes it an elusive target for vaccine design. Analyses of sibling sequence sets (sets of viral sequences derived from one person) show that multiple forms of the immunogenic tip of the loop are found within most HIV-1 infected individuals. Viral V3 sequences obtained from epidemiologically unlinked individuals from North America and Europe show extensive variation. However, some amino acid positions distributed throughout the V3 loop are highly conserved, and there is also conservation of the charge class of amino acid able to occupy certain positions relative to the tip of the loop. By contrast, the sequences obtained from many countries throughout the African continent reveal that V3 is a remarkably fluid region with few absolute constraints on the nature of the amino acids that can occupy most positions in the loop. The high degree of heterogeneity in this region is particularly striking in view of its contribution to biologically important viral functions.

  15. Efficient skin permeation of soluble proteins via flexible and functional nano-carrier.

    PubMed

    Choi, Won Il; Lee, Jong Hyun; Kim, Ja-Young; Kim, Jin-Chul; Kim, Young Ha; Tae, Giyoong

    2012-01-30

    In spite of several intrinsic and distinct advantages, a topical and transdermal administration of drugs has been limited mainly due to very low permeability of drugs across skin. Especially, it is generally regarded that hydrophilic macromolecules such as proteins, peptides, and vaccines cannot penetrate across skin. In this study, we demonstrated that chitosan-conjugated, Pluronic-based nano-carrier (nanogel) can act as an efficient delivery vehicle of hydrophilic proteins across human skin. The functional nano-carrier (<100 nm in size), chemically-crosslinking Pluronic F 127 with chitosan conjugation, is flexible and soft with reservoir characteristics for biomacromolecules. The in-vitro permeation experiments through human cadaver skin revealed remarkable permeability of hydrophilic proteins of various sizes including FITC-BSA (67 kDa) and FITC-Insulin (6 kDa) by direct penetration of the nano-carrier across skin. The bioactivity post-permeation of proteins via the functional nano-carrier was also confirmed by delivering ß-galactosidase. Results presented in this paper suggest the use of chitosan-conjugated flexible nano-carrier as a novel platform for transcutaneous delivery of hydrophilic macromolecules and other drug-delivery applications.

  16. Ultrafast dynamics of nonequilibrium resonance energy transfer and probing globular protein flexibility of myoglobin.

    PubMed

    Stevens, Jeffrey A; Link, Justin J; Zang, Chen; Wang, Lijuan; Zhong, Dongping

    2012-03-22

    Protein structural plasticity is critical to many biological activities and accurate determination of its temporal and spatial fluctuations is challenging and difficult. Here, we report our extensive characterization of global flexibility of a globular heme protein of myoglobin using resonance energy transfer as a molecular ruler. With site-directed mutagenesis, we use a tryptophan scan to examine local structural fluctuations from B to H helices utilizing 10 tryptophan-heme energy transfer pairs with femtosecond resolution. We observed ultrafast resonance energy transfer dynamics by following a nearly single exponential behavior in 10-100 ps, strongly indicating that the globular structure of myoglobin is relatively rigid, with no observable static or slow dynamic conformational heterogeneity. The observation is against our molecular dynamics simulations, which show large local fluctuations and give multiple exponential energy transfer behaviors, suggesting too flexible of the global structure and thus raising a serious issue of the force fields used in simulations. Finally, these ultrafast energy transfer dynamics all occur on the similar time scales of local environmental relaxations (solvation), leading to nonexponential processes caused by energy relaxations, not structural fluctuations. Our analyses of such processes reveal an intrinsic compressed- and/or stretched-exponential behaviors and elucidate the nature of inherent nonequilibrium of ultrafast resonance energy transfer in proteins. This new concept of compressed nonequilibrium transfer dynamics should be applied to all protein studies by time-resolved Förster resonance energy transfer (FRET).

  17. The statistical conformation of a highly flexible protein: small-angle X-ray scattering of S. aureus protein A.

    PubMed

    Capp, Jo A; Hagarman, Andrew; Richardson, David C; Oas, Terrence G

    2014-08-05

    Staphylococcal protein A (SpA) is a multidomain protein consisting of five globular IgG binding domains separated by a conserved six- to nine-residue flexible linker. We collected SAXS data on the N-terminal protein-binding half of SpA (SpA-N) and constructs consisting of one to five domain modules in order to determine statistical conformation of this important S. aureus virulence factor. We fit the SAXS data to a scattering function based on a new polymer physics model, which provides an analytical description of the SpA-N statistical conformation. We describe a protocol for systematically determining the appropriate level of modeling to fit a SAXS data set based on goodness of fit and whether the addition of parameters improves it. In the case of SpA-N, the analytical polymer physics description provides a depiction of the statistical conformation of a flexible protein that, while lacking atomistic detail, properly reflects the information content of the data.

  18. Managing Demand and Capacity Using Multi-Sector Planning and Flexible Airspace: Human-in-the-Loop Evaluation of NextGen

    NASA Technical Reports Server (NTRS)

    Lee, Paul U.; Smith, Nancy M.; Prevot, Thomas; Homola, Jeffrey R.

    2010-01-01

    When demand for an airspace sector exceeds capacity, the balance can be re-established by reducing the demand, increasing the capacity, or both. The Multi-Sector Planner (MSP) concept has been proposed to better manage traffic demand by modifying trajectories across multiple sectors. A complementary approach to MSP, called Flexible Airspace Management (FAM), reconfigures the airspace such that capacity can be reallocated dynamically to balance the traffic demand across multiple sectors, resulting in fewer traffic management initiatives. The two concepts have been evaluated with a series of human-in-the-loop simulations at the Airspace Operations Laboratory to examine and refine the roles of the human operators in these concepts, as well as their tools and procedural requirements. So far MSP and FAM functions have been evaluated individually but the integration of the two functions is desirable since there are significant overlaps in their goals, geographic/temporal scope of the problem space, and the implementation timeframe. Ongoing research is planned to refine the humans roles in the integrated concept.

  19. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Methods in Enzymology: “Flexible backbone sampling methods to model and design protein alternative conformations”

    PubMed Central

    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 remains 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 co-variation 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. PMID:23422426

  1. Origins of Structural Flexibility in Protein-Based Supramolecular Polymers Revealed by DEER Spectroscopy

    PubMed Central

    2015-01-01

    Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron–electron resonance (DEER) spectroscopy. Experimental spin–spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer. PMID:25060334

  2. Docking flexible ligands in proteins with a solvent exposure- and distance-dependent dielectric function

    NASA Astrophysics Data System (ADS)

    Garden, Daniel P.; Zhorov, Boris S.

    2010-02-01

    Physics-based force fields for ligand-protein docking usually determine electrostatic energy with distance-dependent dielectric (DDD) functions, which do not fully account for the dielectric permittivity variance between 2 in the protein core and 80 in bulk water. Here we propose an atom-atom solvent exposure- and distance-dependent dielectric (SEDDD) function, which accounts for both electrostatic and dehydration energy components. Docking was performed using the ZMM program, the AMBER force field, and precomputed libraries of ligand conformers. At the seeding stage, hundreds of thousands of positions and orientations of conformers from the libraries were sampled within the rigid protein. At the refinement stage, the ten lowest-energy structures from the seeding stage were Monte Carlo-minimized with the flexible ligand and flexible protein. A search was considered a success if the root mean square deviation (RMSD) of the ligand atoms in the apparent global minimum from the x-ray structure was <2 Å. Calculations on an examining set of 60 ligand-protein complexes with different DDD functions and solvent-exclusion energy revealed outliers in most of which the ligand-binding site was located at the protein surface. Using a training set of 16 ligand-protein complexes, which did not overlap with the examining set, we parameterized the SEDDD function to minimize the RMSD of the apparent global minima from the x-ray structures. Recalculation of the examining set with the SEDDD function demonstrated a 20% increase in the success rate versus the best-performing DDD function.

  3. Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

    SciTech Connect

    Chen, Hui; Mustafi, Sourajit M.; LeMaster, David M.; Li, Zhong; Héroux, Annie; Li, Hongmin; Hernández, Griselda

    2014-03-01

    Two crystal forms of unligated FKBP12.6 exhibit multiple conformations in the active site and in the 80s loop, the primary site for known protein-recognition interactions. The previously unreported NMR backbone assignment of FKBP12.6 revealed extensive doubling of amide resonances, which reflects a slow conformational transition centered in the 80s loop. The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca{sup 2+} channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2{sub 1} and P3{sub 1}21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3{sub 1}21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition ‘80s loop’ is flipped in the P2{sub 1} crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

  4. Probing Protein Packing Surrounding the Residues In and Flanking the Nicotinic Acetylcholine Receptor M2M3 Loop

    PubMed Central

    Wiltfong, Roger Ernest; Jansen, Michaela

    2009-01-01

    Nicotinic Acetylcholine Receptors (nAChR) are cation-selective, ligand-gated ion channels of the Cys-loop gene superfamily. The recent crystal structure of a bacterial homologue from Erwinia chrysanthemi (ELIC) agrees with previous structures of the N-terminal domain of acetylcholine-binding protein (AChBP) and of the electronmicroscopy derived Torpedo nAChR structure. However, the ELIC transmembrane domain is significantly more tightly packed than the corresponding region of the Torpedo nAChR. We investigated the tightness of protein packing surrounding the extracellular end of the M2 transmembrane segment and around the loop connecting the M2 and M3 segments using the substituted cysteine accessibility method (SCAM). The M2 20′ to 27′ residues were highly water accessible and the variation in reaction rates were consistent with this region being α-helical. At all positions tested, the presence of ACh changed MTSEA modification rates by less than 10-fold. In the presence of ACh, reaction rates for residues in the last extracellular α-helical turn of M2 and in the M2M3 loop increased, whereas rates in M2's penultimate α-helical turn decreased. Only 3 out of 8 M2M3 loop residues were accessible to MTSEA in both the presence and absence of ACh. We infer that the protein packing around the M2M3 loop is tight, consistent with it's location at the interdomain interface where it is involved in the transduction of ligand binding in the extracellular domain to gating in the transmembrane domain. Our data indicate that the Torpedo nAChR transmembrane domain structure is a better model than the ELIC structure for eukaryotic Cys loop receptors. PMID:19211870

  5. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    NASA Astrophysics Data System (ADS)

    Martinez, N.; Michoud, G.; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

    2016-09-01

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

  6. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    PubMed Central

    Martinez, N.; Michoud, G.; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

    2016-01-01

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure. PMID:27595789

  7. Combining conformational flexibility and continuum electrostatics for calculating pK(a)s in proteins.

    PubMed Central

    Georgescu, Roxana E; Alexov, Emil G; Gunner, Marilyn R

    2002-01-01

    Protein stability and function relies on residues being in their appropriate ionization states at physiological pH. In situ residue pK(a)s also provides a sensitive measure of the local protein environment. Multiconformation continuum electrostatics (MCCE) combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side chain ionization and conformation. The response of protein to charges is incorporated both in the protein dielectric constant (epsilon(prot)) of four and by explicit conformational changes. The pK(a) of 166 residues in 12 proteins was determined. The root mean square error is 0.83 pH units, and >90% have errors of <1 pH units whereas only 3% have errors >2 pH units. Similar results are found with crystal and solution structures, showing that the method's explicit conformational sampling reduces sensitivity to the initial structure. The outcome also changes little with protein dielectric constant (epsilon(prot) 4-20). Multiconformation continuum electrostatics titrations show coupling of conformational flexibility and changes in ionization state. Examples are provided where ionizable side chain position (protein G), Asn orientation (lysozyme), His tautomer distribution (RNase A), and phosphate ion binding (RNase A and H) change with pH. Disallowing these motions changes the calculated pK(a). PMID:12324397

  8. The Kl-3 Loop of the Y Chromosome of Drosophila Melanogaster Binds a Tektin-like Protein

    PubMed Central

    Pisano, C.; Bonaccorsi, S.; Gatti, M.

    1993-01-01

    Primary spermatocyte nuclei of Drosophila melanogaster exhibit three giant lampbrush-like loops formed by the kl-5, kl-3 and ks-1 Y-chromosome fertility factors. These structures contain and abundantly transcribe highly repetitive, simple sequence DNAs and accumulate large amounts of non-Y-encoded proteins. By immunizing mice with the 53-kD fraction (enriched in β(2)-tubulin) excised from a sodium dodecyl sulfate-polyacrylamide gel loaded with Drosophila testis proteins we raised a polyclonal antibody, designated as T53-1, which decorates the kl-3 loop and the sperm flagellum. Two dimensional immunoblot analysis showed that the T53-1 antibody reacts with a single protein of about 53 kD, different from the tubulins and present both in X/Y and X/O males. Moreover, the antigen recognized by the T53-1 antibody proved to be testis-specific because it was detected in testes and seminal vesicles but not in other male tissues or in females. The characteristics of the protein recognized by the T53-1 antibody suggested that it might be a member of a class of axonemal proteins, the tektins, known to form Sarkosyl-urea insoluble filaments in the wall of flagellar microtubules. Purification of the Sarkosyl-urea insoluble fraction of D. melanogaster sperm revealed that it contains four polypeptides having molecular masses ranging from 51 to 57 kD. One of these polypeptides reacts strongly with the T53-1 antibody but none of them reacts with antitubulin antibodies. These results indicate that the kl-3 loop binds a non-Y encoded, testis-specific, tektin-like protein which is a constituent of the sperm flagellum. This finding supports the hypothesis that the Y loops fulfill a protein-binding function required for the proper assembly of the axoneme components. PMID:8454204

  9. Full automation and validation of a flexible ELISA platform for host cell protein and protein A impurity detection in biopharmaceuticals.

    PubMed

    Rey, Guillaume; Wendeler, Markus W

    2012-11-01

    Monitoring host cell protein (HCP) and protein A impurities is important to ensure successful development of recombinant antibody drugs. Here, we report the full automation and validation of an ELISA platform on a robotic system that allows the detection of Chinese hamster ovary (CHO) HCPs and residual protein A of in-process control samples and final drug substance. The ELISA setup is designed to serve three main goals: high sample throughput, high quality of results, and sample handling flexibility. The processing of analysis requests, determination of optimal sample dilutions, and calculation of impurity content is performed automatically by a spreadsheet. Up to 48 samples in three unspiked and spiked dilutions each are processed within 24 h. The dilution of each sample is individually prepared based on the drug concentration and the expected impurity content. Adaptable dilution protocols allow the analysis of sample dilutions ranging from 1:2 to 1:2×10(7). The validity of results is assessed by automatic testing for dilutional linearity and spike recovery for each sample. This automated impurity ELISA facilitates multi-project process development, is easily adaptable to other impurity ELISA formats, and increases analytical capacity by combining flexible sample handling with high data quality.

  10. Investigation of the flexibility of protein kinases implicated in the pathology of Alzheimer's disease.

    PubMed

    Mazanetz, Michael P; Laughton, Charles A; Fischer, Peter M

    2014-06-30

    The pathological characteristics of Alzheimer's Disease (AD) have been linked to the activity of three particular kinases--Glycogen Synthase Kinase 3β (GSK3β), Cyclin-Dependent Kinase 5 (CDK5) and Extracellular-signal Regulated Kinase 2 (ERK2). As a consequence, the design of selective, potent and drug-like inhibitors of these kinases is of particular interest. Structure-based design methods are well-established in the development of kinase inhibitors. However, progress in this field is limited by the difficulty in obtaining X-ray crystal structures suitable for drug design and by the inability of this method to resolve highly flexible regions of the protein that are crucial for ligand binding. To address this issue, we have undertaken a study of human protein kinases CDK5/p25, CDK5, ERK2 and GSK3β using both conventional molecular dynamics (MD) and the new Active Site Pressurisation (ASP) methodology, to look for kinase-specific patterns of flexibility that could be leveraged for the design of selective inhibitors. ASP was used to examine the intrinsic flexibility of the ATP-binding pocket for CDK5/p25, CDK5 and GSK3β where it is shown to be capable of inducing significant conformational changes when compared with X-ray crystal structures. The results from these experiments were used to quantify the dynamics of each protein, which supported the observations made from the conventional MD simulations. Additional information was also derived from the ASP simulations, including the shape of the ATP-binding site and the rigidity of the ATP-binding pocket. These observations may be exploited in the design of selective inhibitors of GSK3β, CDK5 and ERK2.

  11. HIV-1 Nucleocapsid Protein Switches the Pathway of TAR RNA/DNA Annealing from Loop-Loop “Kissing” to “Zipper”

    PubMed Central

    Vo, My-Nuong; Barany, George; Rouzina, Ioulia; Musier-Forsyth, Karin

    2009-01-01

    Summary The chaperone activity of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) facilitates multiple nucleic acid rearrangements that are critical for reverse transcription of the single-stranded RNA genome into double-stranded DNA. Annealing of the trans-activation response element (TAR) RNA hairpin to a complementary TAR DNA hairpin is an essential step in the minus-strand transfer step of reverse transcription. Previously, we used truncated 27-nucleotide (nt) mini-TAR RNA and DNA constructs to investigate this annealing reaction pathway in the presence and absence of HIV-1 NC. In this work, full-length 59-nt TAR RNA and TAR DNA constructs were used to systematically study TAR hairpin annealing kinetics. In the absence of NC, full-length TAR hairpin annealing is ∼10-fold slower than mini-TAR annealing. Similar to mini-TAR annealing, the reaction pathway for TAR in the absence of NC involves the fast formation of an unstable “kissing” loop intermediate, followed by a slower conversion to an extended duplex. NC facilitates the annealing of TAR by ∼105-fold by stabilizing the bimolecular intermediate (∼104-fold) and promoting the subsequent exchange reaction (∼10-fold). In contrast to the mini-TAR annealing pathway, wherein NC-mediated annealing can initiate through both loop-loop kissing and a distinct “zipper” pathway involving nucleation at the 3′/5′ terminal ends, full-length TAR hairpin annealing switches predominantly to the zipper pathway in the presence of saturated NC. PMID:19154737

  12. A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus

    SciTech Connect

    Zhang Shuliu; Li Li; Woodson, Sara E.; Huang, Claire Y.-H.; Kinney, Richard M.; Barrett, Alan D.T. ||||; Beasley, David W.C. |||. E-mail: d.beasley@utmb.edu

    2006-09-15

    Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.

  13. Phylogenetic analysis of the human basic helix-loop-helix proteins

    PubMed Central

    2002-01-01

    Background The basic helix-loop-helix (bHLH) proteins are a large and complex multigene family of transcription factors with important roles in animal development, including that of fruitflies, nematodes and vertebrates. The identification of orthologous relationships among the bHLH genes from these widely divergent taxa allows reconstruction of the putative complement of bHLH genes present in the genome of their last common ancestor. Results We identified 39 different bHLH genes in the worm Caenorhabditis elegans, 58 in the fly Drosophila melanogaster and 125 in human (Homo sapiens). We defined 44 orthologous families that include most of these bHLH genes. Of these, 43 include both human and fly and/or worm genes, indicating that genes from these families were already present in the last common ancestor of worm, fly and human. Only two families contain both yeast and animal genes, and no family contains both plant and animal bHLH genes. We suggest that the diversification of bHLH genes is directly linked to the acquisition of multicellularity, and that important diversification of the bHLH repertoire occurred independently in animals and plants. Conclusions As the last common ancestor of worm, fly and human is also that of all bilaterian animals, our analysis indicates that this ancient ancestor must have possessed at least 43 different types of bHLH, highlighting its genomic complexity. PMID:12093377

  14. The Basic Helix-Loop-Helix Protein Family: Comparative Genomics and Phylogenetic Analysis

    PubMed Central

    Ledent, Valérie; Vervoort, Michel

    2001-01-01

    The basic Helix-Loop-Helix (bHLH) proteins are transcription factors that play important roles during the development of various metazoans including fly, nematode, and vertebrates. They are also involved in human diseases, particularly in cancerogenesis. We made an extensive search for bHLH sequences in the completely sequenced genomes of Caenorhabditis elegans and of Drosophila melanogaster. We found 35 and 56 different genes, respectively, which may represent the complete set of bHLH of these organisms. A phylogenetic analysis of these genes, together with a large number (>350) of bHLH from other sources, led us to define 44 orthologous families among which 36 include bHLH from animals only, and two have representatives in both yeasts and animals. In addition, we identified two bHLH motifs present only in yeast, and four that are present only in plants; however, the latter number is certainly an underestimate. Most animal families (35/38) comprise fly, nematode, and vertebrate genes, suggesting that their common ancestor, which lived in pre-Cambrian times (600 million years ago) already owned as many as 35 different bHLH genes. PMID:11337472

  15. Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.

    PubMed

    Hoshijima, K; Kohyama, A; Watakabe, I; Inoue, K; Sakamoto, H; Shimura, Y

    1995-09-11

    Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.

  16. Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins.

    PubMed

    Ganguly, Anindya; Park, Minho; Kesawat, Mahipal Singh; Cho, Hyung-Taeg

    2014-04-01

    Different PIN-FORMED proteins (PINs) contribute to intercellular and intracellular auxin transport, depending on their distinctive subcellular localizations. Arabidopsis thaliana PINs with a long hydrophilic loop (HL) (PIN1 to PIN4 and PIN7; long PINs) localize predominantly to the plasma membrane (PM), whereas short PINs (PIN5 and PIN8) localize predominantly to internal compartments. However, the subcellular localization of the short PINs has been observed mostly for PINs ectopically expressed in different cell types, and the role of the HL in PIN trafficking remains unclear. Here, we tested whether a long PIN-HL can provide its original molecular cues to a short PIN by transplanting the HL. The transplanted long PIN2-HL was sufficient for phosphorylation and PM trafficking of the chimeric PIN5:PIN2-HL but failed to provide the characteristic polarity of PIN2. Unlike previous observations, PIN5 showed clear PM localization in diverse cell types where PIN5 is natively or ectopically expressed and even polar PM localization in one cell type. Furthermore, in the root epidermis, the subcellular localization of PIN5 switched from PM to internal compartments according to the developmental stage. Our results suggest that the long PIN-HL is partially modular for the trafficking behavior of PINs and that the intracellular trafficking of PIN is plastic depending on cell type and developmental stage.

  17. ParaDock: a flexible non-specific DNA--rigid protein docking algorithm.

    PubMed

    Banitt, Itamar; Wolfson, Haim J

    2011-11-01

    Accurate prediction of protein-DNA complexes could provide an important stepping stone towards a thorough comprehension of vital intracellular processes. Few attempts were made to tackle this issue, focusing on binding patch prediction, protein function classification and distance constraints-based docking. We introduce ParaDock: a novel ab initio protein-DNA docking algorithm. ParaDock combines short DNA fragments, which have been rigidly docked to the protein based on geometric complementarity, to create bent planar DNA molecules of arbitrary sequence. Our algorithm was tested on the bound and unbound targets of a protein-DNA benchmark comprised of 47 complexes. With neither addressing protein flexibility, nor applying any refinement procedure, CAPRI acceptable solutions were obtained among the 10 top ranked hypotheses in 83% of the bound complexes, and 70% of the unbound. Without requiring prior knowledge of DNA length and sequence, and within <2 h per target on a standard 2.0 GHz single processor CPU, ParaDock offers a fast ab initio docking solution.

  18. Microscopic insight into role of protein flexibility during ion exchange chromatography by nuclear magnetic resonance and quartz crystal microbalance approaches.

    PubMed

    Hao, Dongxia; Ge, Jia; Huang, Yongdong; Zhao, Lan; Ma, Guanghui; Su, Zhiguo

    2016-03-18

    Driven by the prevalent use of ion exchange chromatography (IEC) for polishing therapeutic proteins, many rules have been formulated to summarize the different dependencies between chromatographic data and various operational parameters of interest based on statically determined interactions. However, the effects of the unfolding of protein structures and conformational stability are not as well understood. This study focuses on how the flexibility of proteins perturbs retention behavior at the molecular scale using microscopic characterization approaches, including hydrogen-deuterium (H/D) exchange detected by NMR and a quartz crystal microbalance (QCM). The results showed that a series of chromatographic retention parameters depended significantly on the adiabatic compressibility and structural flexibility of the protein. That is, softer proteins with higher flexibility tended to have longer retention times and stronger affinities on SP Sepharose adsorbents. Tracing the underlying molecular mechanism using NMR and QCM indicated that an easily unfolded flexible protein with a more compact adsorption layer might contribute to the longer retention time on adsorbents. The use of NMR and QCM provided a previously unreported approach for elucidating the effect of protein structural flexibility on binding in IEC systems.

  19. Conformational flexibility of a human immunoglobulin light chain variable domain by relaxation dispersion nuclear magnetic resonance spectroscopy: implications for protein misfolding and amyloid assembly.

    PubMed

    Mukherjee, Sujoy; Pondaven, Simon P; Jaroniec, Christopher P

    2011-07-05

    The conformational flexibility of a human immunoglobulin κIV light-chain variable domain, LEN, which can undergo conversion to amyloid under destabilizing conditions, was investigated at physiological and acidic pH on a residue-specific basis by multidimensional solution-state nuclear magnetic resonance (NMR) methods. Measurements of backbone chemical shifts and amide (15)N longitudinal and transverse spin relaxation rates and steady-state nuclear Overhauser enhancements indicate that, on the whole, LEN retains its native three-dimensional fold and dimeric state at pH 2 and that the protein backbone exhibits limited fast motions on the picosecond to nanosecond time scale. On the other hand, (15)N Carr--Purcell--Meiboom--Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments of about 5-20 residues and encompassing the N-terminal β-strand and complementarity determining loop regions 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations, in the ~2-4% range. Upon acidification, the minor conformer populations increase significantly, to ~10-15%, with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. These findings provide molecular-level insights about partial protein unfolding at low pH and point to the LEN dimer dissociation, initiated by increased conformational flexibility in several well-defined regions, as being one of the important early events leading to amyloid assembly.

  20. The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast

    PubMed Central

    Aronica, Lucia; Kasparek, Torben; Ruchman, David; Marquez, Yamile; Cipak, Lubos; Cipakova, Ingrid; Anrather, Dorothea; Mikolaskova, Barbora; Radtke, Maximilian; Sarkar, Sovan; Pai, Chen-Chun; Blaikley, Elizabeth; Walker, Carol; Shen, Kuo-Fang; Schroeder, Renee; Barta, Andrea; Forsburg, Susan L.; Humphrey, Timothy C.

    2016-01-01

    The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability. PMID:26682798

  1. Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins

    SciTech Connect

    Hai-Ling Hsu; Lan Huang; Julia Tsou Tsan

    1994-02-01

    Tumor-specific activation of the TAL1 gene is the most common genetic alteration seen in patients with T-cell acute lymphoblastic leukemia. The TAL1 gene products contain the basic helix-loop-helix (bHLH) domain, a protein dimerization and DNA-binding motif common to several known transcription factors. A binding-site selection procedure has now been used to evaluate the DNA recognition properties of TAL1. These studies demonstrate that TAL1 polypeptides do not have intrinsic DNA-binding activity, presumably because of their inability to form bHLH homodimers. However, TAL1 readily interacts with any of the known class A bHLH proteins (E12, E47, E2-2, and HEB) to form heterodimers that bind DNA in a sequence-specific manner. The TAL1 heterodimers preferentially recognize a subset of E-box elements (CANNTG) that can be represented by the consensus sequence AACAGATGGT. This consensus is composed of half-sites for recognition by the participating class A bHLH polypeptide (AACAG) and the TAL1 polypeptide (ATGGT). TAL1 heterodimers with DNA-binding activity are readily detected in nuclear extracts of Jurkat, a leukemic cell line derived from a patient with T-cell acute lymphoblastic leukemia. Hence, TAL1 is likely to bind and regulate the transcription of a unique subset of subordinate target genes, some of which may mediate the malignant function of TAL1 during T-cell leukemogenesis. 48 refs., 10 figs.

  2. Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues.

    PubMed

    Rasila, Tiina S; Vihinen, Mauno; Paulin, Lars; Haapa-Paananen, Saija; Savilahti, Harri

    2012-01-01

    MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

  3. An anchor-dependent molecular docking process for docking small flexible molecules into rigid protein receptors.

    PubMed

    Lin, Thy-Hou; Lin, Guan-Liang

    2008-08-01

    A molecular docking method designated as ADDock, anchor-dependent molecular docking process for docking small flexible molecules into rigid protein receptors, is presented in this article. ADDock makes the bond connection lists for atoms based on anchors chosen for building molecular structures for docking small flexible molecules or ligands into rigid active sites of protein receptors. ADDock employs an extended version of piecewise linear potential for scoring the docked structures. Since no translational motion for small molecules is implemented during the docking process, ADDock searches the best docking result by systematically changing the anchors chosen, which are usually the single-edge connected nodes or terminal hydrogen atoms of ligands. ADDock takes intact ligand structures generated during the docking process for computing the docked scores; therefore, no energy minimization is required in the evaluation phase of docking. The docking accuracy by ADDock for 92 receptor-ligand complexes docked is 91.3%. All these complexes have been docked by other groups using other docking methods. The receptor-ligand steric interaction energies computed by ADDock for some sets of active and inactive compounds selected and docked into the same receptor active sites are apparently separated. These results show that based on the steric interaction energies computed between the docked structures and receptor active sites, ADDock is able to separate active from inactive compounds for both being docked into the same receptor.

  4. The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

    PubMed Central

    Daley, Margaret E.; Sykes, Brian D.

    2003-01-01

    Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand. PMID:12824479

  5. Conformational flexibility and the mechanisms of allosteric transitions in topologically similar proteins

    NASA Astrophysics Data System (ADS)

    Tripathi, Swarnendu; Portman, John J.

    2011-08-01

    Conformational flexibility plays a central role in allosteric transition of proteins. In this paper, we extend the analysis of our previous study [S. Tripathi and J. J. Portman, Proc. Natl. Acad. Sci. U.S.A. 106, 2104 (2009)] to investigate how relatively minor structural changes of the meta-stable states can significantly influence the conformational flexibility and allosteric transition mechanism. We use the allosteric transitions of the domains of calmodulin as an example system to highlight the relationship between the transition mechanism and the inter-residue contacts present in the meta-stable states. In particular, we focus on the origin of transient local unfolding (cracking), a mechanism that can lower free energy barriers of allosteric transitions, in terms of the inter-residue contacts of the meta-stable states and the pattern of local strain that develops during the transition. We find that the magnitude of the local strain in the protein is not the sole factor determining whether a region will ultimately crack during the transition. These results emphasize that the residue interactions found exclusively in one of the two meta-stable states is the key in understanding the mechanism of allosteric conformational change.

  6. Conformational flexibility and the mechanisms of allosteric transitions in topologically similar proteins.

    PubMed

    Tripathi, Swarnendu; Portman, John J

    2011-08-21

    Conformational flexibility plays a central role in allosteric transition of proteins. In this paper, we extend the analysis of our previous study [S. Tripathi and J. J. Portman, Proc. Natl. Acad. Sci. U.S.A. 106, 2104 (2009)] to investigate how relatively minor structural changes of the meta-stable states can significantly influence the conformational flexibility and allosteric transition mechanism. We use the allosteric transitions of the domains of calmodulin as an example system to highlight the relationship between the transition mechanism and the inter-residue contacts present in the meta-stable states. In particular, we focus on the origin of transient local unfolding (cracking), a mechanism that can lower free energy barriers of allosteric transitions, in terms of the inter-residue contacts of the meta-stable states and the pattern of local strain that develops during the transition. We find that the magnitude of the local strain in the protein is not the sole factor determining whether a region will ultimately crack during the transition. These results emphasize that the residue interactions found exclusively in one of the two meta-stable states is the key in understanding the mechanism of allosteric conformational change.

  7. A method for including protein flexibility in protein-ligand docking: improving tools for database mining and virtual screening.

    PubMed

    Broughton, H B

    2000-06-01

    Second-generation methods for docking ligands into their biological receptors, such as FLOG, provide for flexibility of the ligand but not of the receptor. Molecular dynamics based methods, such as free energy perturbation, account for flexibility, solvent effects, etc., but are very time consuming. We combined the use of statistical analysis of conformational samples from short-run protein molecular dynamics with grid-based docking protocols and demonstrated improved performance in two test cases. Our statistical analysis explores the importance of the average strength of a potential interaction with the biological target and optionally applies a weighting depending on the variability in the strength of the interaction seen during dynamics simulation. Using these methods, we improved the num-top-ranked 10% of a database of drug-like molecules, in searches based on the three-dimensional structure of the protein. These methods are able to match the ability of manual docking to assess likely inactivity on steric grounds and indeed to rank order ligands from a homologous series of cyclooxygenase-2 inhibitors with good correlation to their true activity. Furthermore, these methods reduce the need for human intervention in setting up molecular docking experiments.

  8. Influence of Protein Flexibility and Peptide Conformation on Reactivity of Monoclonal Anti-Peptide Antibodies with a Protein α -helix

    NASA Astrophysics Data System (ADS)

    Fieser, Terry M.; Tainer, John A.; Geysen, H. Mario; Houghten, Richard A.; Lerner, Richard A.

    1987-12-01

    Monoclonal antibodies against an α -helical region of the iron-containing, oxygen-binding protein myohemerythrin were isolated following immunization of mice with either the whole protein or a peptide homolog of the helix. Three distinct epitopes within the myohemerythrin helix were identified. The individual residues within two of these epitopes that were essential for antibody binding were determined by measuring antibody binding to a set of peptides in which each amino acid of the epitope was replaced in turn by each of the other 19 amino acids. Hydrophilic residues that are exposed in the native conformation and buried, hydrophobic residues were both shown to be irreplaceable, suggesting their direct involvement in antibody binding. The influence of antigen conformation on antibody binding to these amphipathic epitopes was assessed by measuring the relative affinities of the antibodies for peptides, intact protein, and apoprotein. All of the antibodies bound to apoprotein better than to native protein, indicating that relaxation of the native structure by removal of the iron center increases antibody affinity for myohemerythrin. However, not all of the antibodies tested bound to peptides better than to protein, suggesting that increased antigen flexibility is not always sufficient to maximize antibody binding. Antibody binding to peptides appeared to also be influenced by the ability of the peptides to attain secondary structure at the epitopes, either alone or due to carrier influences.

  9. Gold nanoparticle-embedded silk protein-ZnO nanorod hybrids for flexible bio-photonic devices.

    PubMed

    Gogurla, Narendar; Kundu, Subhas C; Ray, Samit K

    2017-04-07

    Silk protein has been used as a biopolymer substrate for flexible photonic devices. Here, we demonstrate ZnO nanorod array hybrid photodetectors on Au nanoparticle-embedded silk protein for flexible optoelectronics. Hybrid samples exhibit optical absorption at the band edge of ZnO as well as plasmonic energy due to Au nanoparticles, making them attractive for selective UV and visible wavelength detection. The device prepared on Au-silk protein shows a much lower dark current and a higher photo to dark-current ratio of ∼10(5) as compared to the control sample without Au nanoparticles. The hybrid device also exhibits a higher specific detectivity due to higher responsivity arising from the photo-generated hole trapping by Au nanoparticles. Sharp pulses in the transient photocurrent have been observed in devices prepared on glass and Au-silk protein substrates due to the light induced pyroelectric effect of ZnO, enabling the demonstration of self-powered photodetectors at zero bias. Flexible hybrid detectors have been demonstrated on Au-silk/polyethylene terephthalate substrates, exhibiting characteristics similar to those fabricated on rigid glass substrates. A study of the performance of photodetectors with different bending angles indicates very good mechanical stability of silk protein based flexible devices. This novel concept of ZnO nanorod array photodetectors on a natural silk protein platform provides an opportunity to realize integrated flexible and self-powered bio-photonic devices for medical applications in near future.

  10. Gold nanoparticle-embedded silk protein-ZnO nanorod hybrids for flexible bio-photonic devices

    NASA Astrophysics Data System (ADS)

    Gogurla, Narendar; Kundu, Subhas C.; Ray, Samit K.

    2017-04-01

    Silk protein has been used as a biopolymer substrate for flexible photonic devices. Here, we demonstrate ZnO nanorod array hybrid photodetectors on Au nanoparticle-embedded silk protein for flexible optoelectronics. Hybrid samples exhibit optical absorption at the band edge of ZnO as well as plasmonic energy due to Au nanoparticles, making them attractive for selective UV and visible wavelength detection. The device prepared on Au-silk protein shows a much lower dark current and a higher photo to dark-current ratio of ∼105 as compared to the control sample without Au nanoparticles. The hybrid device also exhibits a higher specific detectivity due to higher responsivity arising from the photo-generated hole trapping by Au nanoparticles. Sharp pulses in the transient photocurrent have been observed in devices prepared on glass and Au-silk protein substrates due to the light induced pyroelectric effect of ZnO, enabling the demonstration of self-powered photodetectors at zero bias. Flexible hybrid detectors have been demonstrated on Au-silk/polyethylene terephthalate substrates, exhibiting characteristics similar to those fabricated on rigid glass substrates. A study of the performance of photodetectors with different bending angles indicates very good mechanical stability of silk protein based flexible devices. This novel concept of ZnO nanorod array photodetectors on a natural silk protein platform provides an opportunity to realize integrated flexible and self-powered bio-photonic devices for medical applications in near future.

  11. DNA adenine methyltransferase facilitated diffusion is enhanced by protein-DNA "roadblock" complexes that induce DNA looping.

    PubMed

    Pollak, Adam J; Reich, Norbert O

    2015-04-07

    The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein-DNA complexes (roadblocks) diminish and-interestingly-enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5'-GATC-3') with tightly bound EcoRV ENase-DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam's processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a single DNA molecule using an "intersegmental hopping" mechanism. This mechanism involves the protein hopping between looped DNA segments. Both roadblock systems can cause the DNA to loop and therefore facilitate intersegmental hopping. For Lrp, this only occurs when the Dam sites are separated (by >134bp) such that they can be looped around the protein. Intersegmental hopping may well be a general mechanism for proteins that navigate long distances along compacted DNA. Unlike Dam, EcoRI ENase (recognizes 5'-GAATTC-3') relies extensively on a sliding mechanism, and as expected, Lrp decreases its processivity. Our systematic use of protein roadblocks provides a powerful strategy to differentiate between site location mechanisms.

  12. Functional importance of GGXG sequence motifs in putative reentrant loops of 2HCT and ESS transport proteins.

    PubMed

    Dobrowolski, Adam; Lolkema, Juke S

    2009-08-11

    The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)-citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important for function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was

  13. Characterization of Protein Flexibility Using Small-Angle X-Ray Scattering and Amplified Collective Motion Simulations

    PubMed Central

    Wen, Bin; Peng, Junhui; Zuo, Xiaobing; Gong, Qingguo; Zhang, Zhiyong

    2014-01-01

    Large-scale flexibility within a multidomain protein often plays an important role in its biological function. Despite its inherent low resolution, small-angle x-ray scattering (SAXS) is well suited to investigate protein flexibility and determine, with the help of computational modeling, what kinds of protein conformations would coexist in solution. In this article, we develop a tool that combines SAXS data with a previously developed sampling technique called amplified collective motions (ACM) to elucidate structures of highly dynamic multidomain proteins in solution. We demonstrate the use of this tool in two proteins, bacteriophage T4 lysozyme and tandem WW domains of the formin-binding protein 21. The ACM simulations can sample the conformational space of proteins much more extensively than standard molecular dynamics (MD) simulations. Therefore, conformations generated by ACM are significantly better at reproducing the SAXS data than are those from MD simulations. PMID:25140431

  14. Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties.

    PubMed Central

    Alexov, E G; Gunner, M R

    1997-01-01

    A method for combining calculations of residue pKa's with changes in the position of polar hydrogens has been developed. The Boltzmann distributions of proton positions in hydroxyls and neutral titratable residues are found in the same Monte Carlo sampling procedure that determines the amino acid ionization states at each pH. Electrostatic, Lennard-Jones potentials, and torsion angle energies are considered at each proton position. Many acidic and basic residues are found to have significant electrostatic interactions with either a water- or hydroxyl-containing side chain. Protonation state changes are coupled to reorientation of the neighboring hydroxyl dipoles, resulting in smaller free energy differences between neutral and ionized residues than when the protein is held rigid. Multiconformation pH titration gives better agreement with the experimental pKa's for triclinic hen egg lysozyme than conventional rigid protein calculations. The hydroxyl motion significantly increases the protein dielectric response, making it sensitive to the composition of the local protein structure. More than one conformer per residue is often found at a given pH, providing information about the distribution of low-energy lysozyme structures. Images FIGURE 2 FIGURE 6 PMID:9129810

  15. Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

    PubMed Central

    Chen, Hui; Mustafi, Sourajit M.; LeMaster, David M.; Li, Zhong; Héroux, Annie; Li, Hongmin; Hernández, Griselda

    2014-01-01

    The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca2+ channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P21 and P3121 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3121 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition ‘80s loop’ is flipped in the P21 crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring. PMID:24598733

  16. Regulation of renal hemodynamics after protein feeding: effects of loop diuretics.

    PubMed

    Woods, L L; DeYoung, D R; Smith, B E

    1991-11-01

    These studies were designed to test the hypothesis that the renal vasodilation and increased glomerular filtration rate (GFR) after a high-protein meal are mediated by the tubuloglomerular feedback (TGF) mechanism. In eight chronically instrumented conscious dogs, a meal of raw beef (10 g/kg) caused GFR to increase from 66 +/- 5 to 90 +/- 7 ml/min and effective renal plasma flow (ERPF) to increase from 191 +/- 25 to 281 +/- 24 ml/min, while plasma alpha-amino N levels rose from 4.0 +/- 0.1 to 7.3 +/- 0.6 mg/dl. On another day the dogs were given an infusion of furosemide to block TGF, and fluid and salt losses were continuously replaced. Furosemide alone caused GFR to increase in most animals, although the average change did not reach statistical significance, and ERPF increased by 31%. Sodium excretion rose from 15 +/- 5 to 2,390 +/- 280 mueq/min, and urine flow rose from 1.17 +/- 0.22 to 20.5 +/- 2.4 ml/min. Autoregulatory capability was also abolished (autoregulatory index = 0.87 +/- 0.09 compared with 0.19 +/- 0.05 before furosemide). However, there was no significant change in GFR and ERPF after a subsequent meat meal in dogs receiving furosemide. On another day, some of the dogs were given another loop diuretic, ethacrynic acid, which caused no change in GFR, whereas its effects on ERPF, sodium excretion, and urine output were similar to those of furosemide. There were also no changes in GFR or ERPF after a meat meal during ethacrynic acid administration, despite normal increases in plasma alpha-amino N.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Accelerated flexible protein-ligand docking using Hamiltonian replica exchange with a repulsive biasing potential

    PubMed Central

    Ostermeir, Katja; Zacharias, Martin

    2017-01-01

    A molecular dynamics replica exchange based method has been developed that allows rapid identification of putative ligand binding sites on the surface of biomolecules. The approach employs a set of ambiguity restraints in replica simulations between receptor and ligand that allow close contacts in the reference replica but promotes transient dissociation in higher replicas. This avoids long-lived trapping of the ligand or partner proteins at nonspecific, sticky, sites on the receptor molecule and results in accelerated exploration of the possible binding regions. In contrast to common docking methods that require knowledge of the binding site, exclude solvent and often keep parts of receptor and ligand rigid the approach allows for full flexibility of binding partners. Application to peptide-protein, protein-protein and a drug-receptor system indicate rapid sampling of near-native binding regions even in case of starting far away from the native binding site outperforming continuous MD simulations. An application on a DNA minor groove binding ligand in complex with DNA demonstrates that it can also be used in explicit solvent simulations. PMID:28207811

  18. Using least median of squares for structural superposition of flexible proteins

    PubMed Central

    Liu, Yu-Shen; Fang, Yi; Ramani, Karthik

    2009-01-01

    Background The conventional superposition methods use an ordinary least squares (LS) fit for structural comparison of two different conformations of the same protein. The main problem of the LS fit that it is sensitive to outliers, i.e. large displacements of the original structures superimposed. Results To overcome this problem, we present a new algorithm to overlap two protein conformations by their atomic coordinates using a robust statistics technique: least median of squares (LMS). In order to effectively approximate the LMS optimization, the forward search technique is utilized. Our algorithm can automatically detect and superimpose the rigid core regions of two conformations with small or large displacements. In contrast, most existing superposition techniques strongly depend on the initial LS estimating for the entire atom sets of proteins. They may fail on structural superposition of two conformations with large displacements. The presented LMS fit can be considered as an alternative and complementary tool for structural superposition. Conclusion The proposed algorithm is robust and does not require any prior knowledge of the flexible regions. Furthermore, we show that the LMS fit can be extended to multiple level superposition between two conformations with several rigid domains. Our fit tool has produced successful superpositions when applied to proteins for which two conformations are known. The binary executable program for Windows platform, tested examples, and database are available from . PMID:19159484

  19. Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

    PubMed

    Pessler, Frank; Hernandez, Nouria

    2003-08-01

    POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

  20. Maximum occurrence analysis of protein conformations for different distributions of paramagnetic metal ions within flexible two-domain proteins.

    PubMed

    Luchinat, Claudio; Nagulapalli, Malini; Parigi, Giacomo; Sgheri, Luca

    2012-02-01

    Multidomain proteins are composed of rigid domains connected by (flexible) linkers. Therefore, the domains may experience a large degree of reciprocal reorientation. Pseudocontact shifts and residual dipolar couplings arising from one or more paramagnetic metals successively placed in a single metal binding site in the protein can be used as restraints to assess the degree of mobility of the different domains. They can be used to determine the maximum occurrence (MO) of each possible protein conformation, i.e. the maximum weight that such conformations can have independently of the real structural ensemble, in agreement with the provided restraints. In the case of two-domain proteins, the metal ions can be placed all in the same domain, or distributed between the two domains. It has been demonstrated that the quantity of independent information for the characterization of the system is larger when all metals are bound in the same domain. At the same time, it has been shown that there are practical advantages in placing the metals in different domains. Here, it is shown that distributing the metals between the domains provides a tool for defining a coefficient of compatibility among the restraints obtained from different metals, without a significant decrease of the capability of the MO values to discriminate among conformations with different weights.

  1. Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a Gram-negative bacterium

    PubMed Central

    Xiong, Xiaoli; Bromley, Elizabeth H. C.; Oelschlaeger, Peter; Woolfson, Derek N.; Spencer, James

    2011-01-01

    Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in Gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for Gram-negative Qnr proteins. The AhQnr crystal structure, the first for any Gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of Gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A ‘tower’ and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in Gram-negative bacteria. PMID:21227918

  2. Predicting protein conformational changes for unbound and homology docking: learning from intrinsic and induced flexibility.

    PubMed

    Chen, Haoran; Sun, Yuanfei; Shen, Yang

    2017-03-01

    Predicting protein conformational changes from unbound structures or even homology models to bound structures remains a critical challenge for protein docking. Here we present a study directly addressing the challenge by reducing the dimensionality and narrowing the range of the corresponding conformational space. The study builds on cNMA-our new framework of partner- and contact-specific normal mode analysis that exploits encounter complexes and considers both intrinsic and induced flexibility. First, we established over a CAPRI (Critical Assessment of PRedicted Interactions) target set that the direction of conformational changes from unbound structures and homology models can be reproduced to a great extent by a small set of cNMA modes. In particular, homology-to-bound interface root-mean-square deviation (iRMSD) can be reduced by 40% on average with the slowest 30 modes. Second, we developed novel and interpretable features from cNMA and used various machine learning approaches to predict the extent of conformational changes. The models learned from a set of unbound-to-bound conformational changes could predict the actual extent of iRMSD with errors around 0.6 Å for unbound proteins in a held-out benchmark subset, around 0.8 Å for unbound proteins in the CAPRI set, and around 1 Å even for homology models in the CAPRI set. Our results shed new insights into origins of conformational differences between homology models and bound structures and provide new support for the low-dimensionality of conformational adjustment during protein associations. The results also provide new tools for ensemble generation and conformational sampling in unbound and homology docking. Proteins 2017; 85:544-556. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Structure and flexibility of the thermophilic cold-shock protein of Thermus aquaticus.

    PubMed

    Jin, Bonghwan; Jeong, Ki-Woong; Kim, Yangmee

    2014-08-29

    The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein (Ta-Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta-Csp has a five-stranded β-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76 °C) of Ta-Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the β1-β2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674 s(-1). The results imply that thermophilic Ta-Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts.

  4. Prediction of HIV-1 protease inhibitor resistance using a protein-inhibitor flexible docking approach.

    PubMed

    Jenwitheesuk, Ekachai; Samudrala, Ram

    2005-01-01

    Emergence of drug resistance remains one of the most challenging issues in the treatment of HIV-1 infection. Here we focus on resistance to HIV-1 protease inhibitors (PIs) at a molecular level, which can be analysed genotypically or phenotypically. Genotypic assays are based on the analysis of mutations associated with reduced drug susceptibility, but are problematic because of the numerous mutations and mutational patterns that confer drug resistance. Phenotypic resistance or susceptibility can be experimentally evaluated by measuring the amount of free drug bound to HIV-1 protease molecules, but this procedure is expensive and time-consuming. To overcome these problems, we have developed a docking protocol that takes protein-inhibitor flexibility into account to predict phenotypic drug resistance. For six FDA-approved Pls and a total of 1792 HIV-1 protease sequence mutants, we used a combination of inhibitor flexible docking and molecular dynamics (MD) simulations to calculate protein-inhibitor binding energies. Prediction results were expressed as fold changes of the calculated inhibitory constant (Ki), and the samples predicted to have fold-increase in calculated Ki above the fixed cut-off were defined as drug resistant. Our combined docking and MD protocol achieved accuracies ranging from 72-83% in predicting resistance/susceptibility for five of the six drugs evaluated. Evaluating the method only on samples where our predictions concurred with established knowledge-based methods resulted in increased accuracies of 83-94% for the six drugs. The results suggest that a physics-based approach, which is readily applicable to any novel PI and/or mutant, can be used judiciously with knowledge-based approaches that require experimental training data to devise accurate models of HIV-1 Pl resistance prediction.

  5. Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

    PubMed Central

    Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

    2014-01-01

    The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

  6. Influence of rotational energy barriers to the conformational search of protein loops in molecular dynamics and ranking the conformations.

    PubMed

    Tappura, K

    2001-08-15

    An adjustable-barrier dihedral angle potential was added as an extension to a novel, previously presented soft-core potential to study its contribution to the efficacy of the search of the conformational space in molecular dynamics. As opposed to the conventional soft-core potential functions, the leading principle in the design of the new soft-core potential, as well as of its extension, the soft-core and adjustable-barrier dihedral angle (SCADA) potential (referred as the SCADA potential), was to maintain the main equilibrium properties of the original force field. This qualifies the methods for a variety of a priori modeling problems without need for additional restraints typically required with the conventional soft-core potentials. In the present study, the different potential energy functions are applied to the problem of predicting loop conformations in proteins. Comparison of the performance of the soft-core and SCADA potential showed that the main hurdles for the efficient sampling of the conformational space of (loops in) proteins are related to the high-energy barriers caused by the Lennard-Jones and Coulombic energy terms, and not to the rotational barriers, although the conformational search can be further enhanced by lowering the rotational barriers of the dihedral angles. Finally, different evaluation methods were studied and a few promising criteria found to distinguish the near-native loop conformations from the wrong ones.

  7. Biological activity of natural flavonoids as impacted by protein flexibility: an example of flavanones.

    PubMed

    Ding, Fei; Peng, Wei

    2015-04-01

    Naturally multifunctional Rutaceae hesperidin and its aglycone hesperetin have a great variety of biopharmaceutical activities, e.g. anti-cancer, anti-inflammatory, antioxidant and antitumor; however, the influence of the molecular structures of hesperidin and hesperetin, and in particular, the structural properties such as flexibility and dynamic features of protein on the biological activities of these bioactive compounds remains ambiguous. In the present study, the biomolecular recognition of crucial biopolymer - albumin from human serum (HSA) with Rutaceae, the recognition differences between HSA-hesperidin and HSA-hesperetin, the key elements that lead to the discrepancies as well as the structural characters of protein to the recognition processes were comparatively examined by employing biophysical approaches at the molecular scale. The results illustrated distinctly that (1) aglycone hesperetin can form stronger noncovalent bonds with HSA and possess higher recognition stability as compared with hesperidin. This phenomenon suggest that the introduction of glycoside structure into flavanone may possibly not be able to increase the noncovalent recognition of flavanone by a biopolymer, and conversely, this event will probably decrease the recognition capacity. (2) Although hesperidin and hesperetin can be located within subdomains IIA and IIIA, respectively, the conformational stability of flavanones in subdomain IIA is greater than subdomain IIIA; as a result, the recognition ability of subdomain IIIA with flavanones is patently lesser than subdomain IIA. These discrepancies likely originate from the unique characteristics of the respective cavity, or more specifically, subdomain IIA is basically a closed space, whereas subdomain IIIA is a semi-open region. Meanwhile, the detailed analyses of root-mean-square fluctuation interpreted the recognition of flavanones by subdomain IIA on HSA, which would evoke larger conformational alterations in several amino acid

  8. Role of Molecular Flexibility and Colloidal Descriptions of Proteins in Crowded Environments from Small-Angle Scattering.

    PubMed

    Castellanos, Maria Monica; Clark, Nicholas J; Watson, Max C; Krueger, Susan; McAuley, Arnold; Curtis, Joseph E

    2016-12-15

    Small-angle scattering is a powerful technique to study molecular conformation and interactions of proteins in solution and in amorphous solids. We have investigated the role of multiple protein configurations in the interaction parameters derived from small-angle scattering for proteins in concentrated solutions. In order to account for the wide configurational space sampled by proteins, we generate ensembles of atomistic structures for lysozyme and monoclonal antibodies, representing globular and flexible proteins, respectively. While recent work has argued that a colloidal approach is inadequate to model proteins, because of the large configurational space that they sample in solution, we find a range of length scales where colloidal models can be used to describe solution scattering data while simultaneously accounting for structural flexibility. We provide insights to determine the length scales where isotropic colloidal models can be used, and find smoothly varying sets of interaction parameters that encompass ensembles of structures. This approach may play an important role in the definition of long-range interactions in coarse-grained models of flexible proteins with experimental scattering constraints. Additionally, we apply the decoupling approximation to ensembles of lysozyme structures with atomistic detail and observe remarkably different results when using geometric solids, such as ellipsoids. The insights from this study provide guidelines for the analysis of small-angle scattering profiles of proteins in crowded environments.

  9. HIV-1 p6 - a structured to flexible multifunctional membrane-interacting protein.

    PubMed

    Solbak, Sara Marie Øie; Reksten, Tove Ragna; Hahn, Friedrich; Wray, Victor; Henklein, Petra; Henklein, Peter; Halskau, Øyvind; Schubert, Ulrich; Fossen, Torgils

    2013-02-01

    The human immunodeficiency virus type 1 (HIV-1) p6 protein has recently been recognized as a docking site for several cellular and viral binding partners and is important for the formation of infectious viruses. Most of its known functions are suggested to occur under hydrophobic conditions near the cytoplasmic membrane, where the protein is presumed to exist in its most structured state. Although p6 is involved in manifold specific interactions, the protein has previously been considered to possess a random structure in aqueous solution. We show that p6 exhibits a defined structure with N- and C-terminal helical domains, connected by a flexible hinge region in 100mM dodecylphosphocholine micelle solution at pH 7 devoid of any organic co-solvents, indicating that this is a genuine limiting structural feature of the molecule in a hydrophobic environment. Furthermore, we show that p6 directly interacts with a cytoplasmic model membrane through both N-terminal and C-terminal regions by use of surface plasmon resonance (SPR) spectroscopy. Phosphorylation of Ser-40 located in the center of the C-terminal α-helix does not alter the secondary structure of the protein but amplifies the interaction with membranes significantly, indicating that p6 binds to the polar head groups at the surface of the cytoplasmic membrane. The increased hydrophobic membrane interaction of p6(23-52) S40F correlated with the observed increased amount of the polyprotein Gag in the RIPA insoluble fraction when Ser40 of p6 was mutated with Phe indicating that p6 modulates the membrane interactions of HIV-1 Gag.

  10. Entropy and free energy of a mobile protein loop in explicit water.

    PubMed

    Cheluvaraja, Srinath; Mihailescu, Mihail; Meirovitch, Hagai

    2008-08-07

    Estimation of the energy from a given Boltzmann sample is straightforward since one just has to average the contribution of the individual configurations. On the other hand, calculation of the absolute entropy, S (hence the absolute free energy F) is difficult because it depends on the entire (unknown) ensemble. We have developed a new method called "the hypothetical scanning molecular dynamics" (HSMD) for calculating the absolute S from a given sample (generated by any simulation technique). In other words, S (like the energy) is "written" on the sample configurations, where HSMD provides a prescription of how to "read" it. In practice, each sample conformation, i, is reconstructed with transition probabilities, and their product leads to the probability of i, hence to the entropy. HSMD is an exact method where all interactions are considered, and the only approximation is due to insufficient sampling. In previous studies HSMD (and HS Monte CarloHSMC) has been extended systematically to systems of increasing complexity, where the most recent is the seven-residue mobile loop, 304-310 (Gly-His-Gly-Ala-Gly-Gly-Ser) of the enzyme porcine pancreatic alpha-amylase modeled by the AMBER force field and AMBER with the implicit solvation GB/SA (paper I, Cheluvaraja, S.; Meirovitch, H. J. Chem. Theory Comput. 2008, 4, 192). In the present paper we make a step further and extend HSMD to the same loop capped with TIP3P explicit water at 300 K. As in paper I, we are mainly interested in entropy and free energy differences between the free and bound microstates of the loop, which are obtained from two separate MD samples of these microstates. The contribution of the loop to S and F is calculated by HSMD and that of water by a particular thermodynamic integration procedure. As expected, the free microstate is more stable than the bound microstate by a total free energy difference, Ffree-Fbound=-4.8+/-1, as compared to -25.5 kcal/mol obtained with GB/SA. We find that relatively

  11. Enhancement of surface nonwettability by grafting loops.

    PubMed

    Pei, Han-Wen; Liu, Xiao-Li; Liu, Hong; Zhu, You-Liang; Lu, Zhong-Yuan

    2017-02-08

    We present a computer simulation study on the nonwettability of a flat surface tethered with deformable looped polymer chains. Two kinds of loops are studied: monodispersed loops (loops with the same length) and polydispersed loops (loops with different lengths). Both kinds of loops include two arrangements: with regularly tethered sites and with randomly tethered sites. Regularly grafted loops form typical grooves on the surface, while randomly grafted loops form a more rugged surface. For monodispersed loops, we analyze the factors that influence the nonwettability when varying the rigidity of the loops. The loops are divided into two categories based on their rigidity according to our previous analysis procedure (Phys. Chem. Chem. Phys., 2016, 18, 18767-18775): rigid loops and flexible loops. It is found that the loop can partially form a re-entrant-like structure, which is helpful to increase the nonwettability of the surface. The surfaces with grafted loops have increased nonwettability, especially those grafted with flexible chains. However, the contact angle on the loop structure cannot further increase for the rigid chains due to a large top layer density (Phys. Chem. Chem. Phys., 2016, 18, 18767-18775). For polydispersed loops, the contact angle is highly related to the rigidity of the long loops that contact the droplet. Different from monodispersed loops, the mechanism of the nonwettability of polydispersed loops is attributed to the supporting ability (rigidity) of long loops.

  12. Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

    PubMed Central

    Burns, C C; Poss, M L; Thomas, E; Overbaugh, J

    1995-01-01

    A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity. PMID:7884859

  13. Structural flexibility allows the functional diversity of potyvirus genome-linked protein VPg.

    PubMed

    Rantalainen, Kimmo I; Eskelin, Katri; Tompa, Peter; Mäkinen, Kristiina

    2011-03-01

    Several viral genome-linked proteins (VPgs) of plant viruses are intrinsically disordered and undergo folding transitions in the presence of partners. This property has been postulated to be one of the factors that enable the functional diversity of the protein. We created a homology model of Potato virus A VPg and positioned the known functions and structural properties of potyviral VPgs on the novel structural model. The model suggests an elongated structure with a hydrophobic core composed of antiparallel β-sheets surrounded by helices and a positively charged contact surface where most of the known activities are localized. The model most probably represents the fold induced immediately after binding of VPg to a negatively charged lipid surface or to SDS. When the charge of the positive surface was lowered by lysine mutations, the efficiencies of in vitro NTP binding, uridylylation reaction, and unspecific RNA binding were reduced and in vivo the infectivity was debilitated. The most likely uridylylation site, Tyr63, locates to the positively charged surface. Surprisingly, a Tyr63Ala mutation did not prevent replication completely but blocked spreading of the virus. Based on the localization of Tyr119 in the model, it was hypothesized to serve as an alternative uridylylation site. Evidence to support the role of Tyr119 in replication was obtained which gives a positive example of the prediction power of the model. Taken together, our experimental data support the features presented in the model and the idea that the functional diversity is attributable to structural flexibility.

  14. Flexible method for conjugation of phenolic lignin model compounds to carrier proteins

    DOE PAGES

    Gao, Ruili; Lu, Fachuang; Zhu, Yimin; ...

    2016-10-03

    Linking lignin model compounds to carrier proteins is required either to raise antibodies to them or to structurally screen antibodies raised against lignins or models. This paper describes a flexible method to link phenolic compounds of interest to cationic bovine serum albumin (cBSA) without interfering with their important structural features. With the guaiacylglycerol- β-guaiacyl ether dimer, for example, the linking was accomplished in 89% yield with the number of dimers per carrier protein being as high as 50; NMR experiments on a 15N- and 13C-labeled conjugation product indicated that 13 dimers were added to the native lysine residues and themore » remainder (~37) to the amine moieties on the ethylenediamine linkers added to BSA; ~32% of the available primary amine groups on cBSA were therefore conjugated to the hapten. As a result, this loading is suitable for attempting to raise new antibodies to plant lignins and for screening.« less

  15. Structural Flexibility Allows the Functional Diversity of Potyvirus Genome-Linked Protein VPg▿ §

    PubMed Central

    Rantalainen, Kimmo I.; Eskelin, Katri; Tompa, Peter; Mäkinen, Kristiina

    2011-01-01

    Several viral genome-linked proteins (VPgs) of plant viruses are intrinsically disordered and undergo folding transitions in the presence of partners. This property has been postulated to be one of the factors that enable the functional diversity of the protein. We created a homology model of Potato virus A VPg and positioned the known functions and structural properties of potyviral VPgs on the novel structural model. The model suggests an elongated structure with a hydrophobic core composed of antiparallel β-sheets surrounded by helices and a positively charged contact surface where most of the known activities are localized. The model most probably represents the fold induced immediately after binding of VPg to a negatively charged lipid surface or to SDS. When the charge of the positive surface was lowered by lysine mutations, the efficiencies of in vitro NTP binding, uridylylation reaction, and unspecific RNA binding were reduced and in vivo the infectivity was debilitated. The most likely uridylylation site, Tyr63, locates to the positively charged surface. Surprisingly, a Tyr63Ala mutation did not prevent replication completely but blocked spreading of the virus. Based on the localization of Tyr119 in the model, it was hypothesized to serve as an alternative uridylylation site. Evidence to support the role of Tyr119 in replication was obtained which gives a positive example of the prediction power of the model. Taken together, our experimental data support the features presented in the model and the idea that the functional diversity is attributable to structural flexibility. PMID:21177813

  16. Interaction of sweet proteins with their receptor. A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin.

    PubMed

    Tancredi, Teodorico; Pastore, Annalisa; Salvadori, Severo; Esposito, Veronica; Temussi, Piero A

    2004-06-01

    The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of "sweet fingers", i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential "sweet fingers" of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNLQC(37-47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the "wedge model", recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett.526, 1-3.].

  17. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins.

    PubMed

    Kikhney, Alexey G; Svergun, Dmitri I

    2015-09-14

    Small-angle X-ray scattering (SAXS) is a biophysical method to study the overall shape and structural transitions of biological macromolecules in solution. SAXS provides low resolution information on the shape, conformation and assembly state of proteins, nucleic acids and various macromolecular complexes. The technique also offers powerful means for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs). Here, the basic principles of SAXS are presented, and profits and pitfalls of the characterization of multidomain flexible proteins and IDPs using SAXS are discussed from the practical point of view. Examples of the synergistic use of SAXS with high resolution methods like X-ray crystallography and nuclear magnetic resonance (NMR), as well as other experimental and in silico techniques to characterize completely, or partially unstructured proteins, are presented.

  18. 'Something in the way she moves': The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI).

    PubMed

    Freedman, Robert B; Desmond, Jasmine L; Byrne, Lee J; Heal, Jack W; Howard, Mark J; Sanghera, Narinder; Walker, Kelly L; Wallis, A Katrine; Wells, Stephen A; Williamson, Richard A; Römer, Rudolf A

    2017-11-01

    Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI. Copyright © 2017. Published by Elsevier B.V.

  19. An Ancient P-Loop GTPase in Rice Is Regulated by a Higher Plant-specific Regulatory Protein*

    PubMed Central

    Cheung, Ming-Yan; Xue, Yan; Zhou, Liang; Li, Man-Wah; Sun, Samuel Sai-Ming; Lam, Hon-Ming

    2010-01-01

    YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein. PMID:20876569

  20. Mining flexible-receptor docking experiments to select promising protein receptor snapshots

    PubMed Central

    2010-01-01

    Background Molecular docking simulation is the Rational Drug Design (RDD) step that investigates the affinity between protein receptors and ligands. Typically, molecular docking algorithms consider receptors as rigid bodies. Receptors are, however, intrinsically flexible in the cellular environment. The use of a time series of receptor conformations is an approach to explore its flexibility in molecular docking computer simulations, but it is extensively time-consuming. Hence, selection of the most promising conformations can accelerate docking experiments and, consequently, the RDD efforts. Results We previously docked four ligands (NADH, TCL, PIF and ETH) to 3,100 conformations of the InhA receptor from M. tuberculosis. Based on the receptor residues-ligand distances we preprocessed all docking results to generate appropriate input to mine data. Data preprocessing was done by calculating the shortest interatomic distances between the ligand and the receptor’s residues for each docking result. They were the predictive attributes. The target attribute was the estimated free-energy of binding (FEB) value calculated by the AutodDock3.0.5 software. The mining inputs were submitted to the M5P model tree algorithm. It resulted in short and understandable trees. On the basis of the correlation values, for NADH, TCL and PIF we obtained more than 95% correlation while for ETH, only about 60%. Post processing the generated model trees for each of its linear models (LMs), we calculated the average FEB for their associated instances. From these values we considered a LM as representative if its average FEB was smaller than or equal the average FEB of the test set. The instances in the selected LMs were considered the most promising snapshots. It totalized 1,521, 1,780, 2,085 and 902 snapshots, for NADH, TCL, PIF and ETH respectively. Conclusions By post processing the generated model trees we were able to propose a criterion of selection of linear models which, in turn, is

  1. Surprising flexibility of leader RNA determinants for r-protein L4-mediated transcription termination in the Escherichia coil S10 operon.

    PubMed

    Zengel, Janice M; Sha, Yizhong; Lindahl, Lasse

    2002-05-01

    Escherichia coli ribosomal protein L4 autogenously regulates transcription of the S10 operon, which encodes L4 and 10 other ribosomal proteins. Regulation results from L4-stimulated premature transcription termination at a U-rich site in the untranslated leader. The process requires transcription factor NusA. Here we report a detailed analysis of the RNA requirements for NusA-dependent, L4-mediated transcription control. We found that efficient regulation requires multiple features of the S10 leader, including two hairpins, called HD and upper HE, a connecting tether, and a U-rich sequence at the distal side of HE. As expected, regulation was optimal when all 7 Us were maintained in the U4CGU3 sequence at the termination site. However, despite the apparent specificity of L4 action on only the S10 operon, there is surprising flexibility at the primary sequence level for the HD-tether-HE region. Changes in the sequence of non-base-paired nucleotides flanking the HD hairpin or an A at the second position of the HD loop reduced L4 regulation, but other changes had little or no effect. Furthermore, generic hairpins from other RNAs could replace the natural HD and upper HE hairpins with little or no reduction of L4 control, suggesting that the secondary structure elements are also relatively generic. The lack of specific sequence requirements suggests that L4 may recognize multiple elements within this region of the nascent leader.

  2. Surprising flexibility of leader RNA determinants for r-protein L4-mediated transcription termination in the Escherichia coil S10 operon.

    PubMed Central

    Zengel, Janice M; Sha, Yizhong; Lindahl, Lasse

    2002-01-01

    Escherichia coli ribosomal protein L4 autogenously regulates transcription of the S10 operon, which encodes L4 and 10 other ribosomal proteins. Regulation results from L4-stimulated premature transcription termination at a U-rich site in the untranslated leader. The process requires transcription factor NusA. Here we report a detailed analysis of the RNA requirements for NusA-dependent, L4-mediated transcription control. We found that efficient regulation requires multiple features of the S10 leader, including two hairpins, called HD and upper HE, a connecting tether, and a U-rich sequence at the distal side of HE. As expected, regulation was optimal when all 7 Us were maintained in the U4CGU3 sequence at the termination site. However, despite the apparent specificity of L4 action on only the S10 operon, there is surprising flexibility at the primary sequence level for the HD-tether-HE region. Changes in the sequence of non-base-paired nucleotides flanking the HD hairpin or an A at the second position of the HD loop reduced L4 regulation, but other changes had little or no effect. Furthermore, generic hairpins from other RNAs could replace the natural HD and upper HE hairpins with little or no reduction of L4 control, suggesting that the secondary structure elements are also relatively generic. The lack of specific sequence requirements suggests that L4 may recognize multiple elements within this region of the nascent leader. PMID:12022224

  3. BP-Dock: a flexible docking scheme for exploring protein-ligand interactions based on unbound structures.

    PubMed

    Bolia, Ashini; Gerek, Z Nevin; Ozkan, S Banu

    2014-03-24

    Molecular docking serves as an important tool in modeling protein-ligand interactions. However, it is still challenging to incorporate overall receptor flexibility, especially backbone flexibility, in docking due to the large conformational space that needs to be sampled. To overcome this problem, we developed a novel flexible docking approach, BP-Dock (Backbone Perturbation-Dock) that can integrate both backbone and side chain conformational changes induced by ligand binding through a multi-scale approach. In the BP-Dock method, we mimic the nature of binding-induced events as a first-order approximation by perturbing the residues along the protein chain with a small Brownian kick one at a time. The response fluctuation profile of the chain upon these perturbations is computed using the perturbation response scanning method. These response fluctuation profiles are then used to generate binding-induced multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, we applied our approach on a large and diverse data set using unbound structures as receptors. We also compared the BP-Dock results with bound and unbound docking, where overall receptor flexibility was not taken into account. Our results highlight the importance of modeling backbone flexibility in docking for recapitulating the experimental binding affinities, especially when an unbound structure is used. With BP-Dock, we can generate a wide range of binding site conformations realized in nature even in the absence of a ligand that can help us to improve the accuracy of unbound docking. We expect that our fast and efficient flexible docking approach may further aid in our understanding of protein-ligand interactions as well as virtual screening of novel targets for rational drug design.

  4. Intrinsic protein flexibility in regulation of cell proliferation: advantages for signaling and opportunities for novel therapeutics.

    PubMed

    Follis, Ariele Viacava; Galea, Charles A; Kriwacki, Richard W

    2012-01-01

    It is now widely recognized that intrinsically disordered (or unstructured) proteins (IDPs, or IUPs) are found in organisms from all kingdoms of life. In eukaryotes, IDPs are highly abundant and perform a wide range of biological functions, including regulation and signaling. Despite increased interest in understanding the structural biology of IDPs, questions remain regarding the mechanisms through which disordered proteins perform their biological function(s). In other words, what are the relationships between disorder and function for IDPs? Several excellent reviews have recently been published that discuss the structural properties of IDPs.1-3 Here, we discuss two IDP systems which illustrate features of dynamic complexes. In the first section, we discuss two IDPs, p21 and p27, which regulate the mammalian cell division cycle by inhibiting cyclin-dependent kinases (Cdks). In the second section, we discuss recent results from Follis, Hammoudeh, Metallo and coworkers demonstrating that the IDP Myc can be bound and inhibited by small molecules through formation of dynamic complexes. Previous studies have shown that polypeptide segments of p21 and p27 are partially folded in isolation and fold further upon binding their biological targets. Interestingly, some portions of p27 which bind to and inhibit Cdk2/cyclin A remain flexible in the bound complex. This residual flexibility allows otherwise buried tyrosine residues within p27 to be phosphorylated by nonreceptor tyrosine kinases (NRTKs). Tyrosine phosphorylation relieves kinase inhibition, triggering Cdk2-mediated phosphorylation of a threonine residue within the flexible C-terminus of p27. This, in turn, marks p27 for ubiquitination and proteasomal degradation, unleashing full Cdk2 activity which drives cell cycle progression. p27, thus, constitutes a conduit for transmission of proliferative signals via posttranslational modifications. Importantly, activation of the p27 signaling conduit by oncogenic NRTKs

  5. Dynameomics: data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction.

    PubMed

    Rysavy, Steven J; Beck, David A C; Daggett, Valerie

    2014-11-01

    Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼ 25-75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. © 2014 The Protein Society.

  6. Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel.

    PubMed

    Parthasarathy, Krupakar; Ng, Lifang; Lin, Xin; Liu, Ding Xiang; Pervushin, Konstantin; Gong, Xiandi; Torres, Jaume

    2008-09-15

    Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane alpha-helical bundles, related by a approximately 50 degrees rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM alpha-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric alpha-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function.

  7. Structural Flexibility of the Pentameric SARS Coronavirus Envelope Protein Ion Channel

    PubMed Central

    Parthasarathy, Krupakar; Ng, Lifang; Lin, Xin; Liu, Ding Xiang; Pervushin, Konstantin; Gong, Xiandi; Torres, Jaume

    2008-01-01

    Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane α-helical bundles, related by a ∼50° rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM α-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric α-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function. PMID:18658207

  8. Adolescent binge ethanol treatment alters adult brain regional volumes, cortical extracellular matrix protein and behavioral flexibility.

    PubMed

    Coleman, Leon Garland; Liu, Wen; Oguz, Ipek; Styner, Martin; Crews, Fulton T

    2014-01-01

    Adolescents binge drink more than any other age group, increasing risk of disrupting the development of the frontal cortex. We hypothesized that adolescent binge drinking would lead to persistent alterations in adulthood. In this study, we modeled adolescent weekend underage binge-drinking, using adolescent mice (post-natal days [P] 28-37). The adolescent intermittent binge ethanol (AIE) treatment includes 6 binge intragastric doses of ethanol in an intermittent pattern across adolescence. Assessments were conducted in adulthood following extended abstinence to determine if there were persistent changes in adults. Reversal learning, open field and other behavioral assessments as well as brain structure using magnetic imaging and immunohistochemistry were determined. We found that AIE did not impact adult Barnes Maze learning. However, AIE did cause reversal learning deficits in adults. AIE also caused structural changes in the adult brain. AIE was associated with adulthood volume enlargements in specific brain regions without changes in total brain volume. Enlarged regions included the orbitofrontal cortex (OFC, 4%), cerebellum (4.5%), thalamus (2%), internal capsule (10%) and genu of the corpus callosum (7%). The enlarged OFC volume in adults after AIE is consistent with previous imaging studies in human adolescents. AIE treatment was associated with significant increases in the expression of several extracellular matrix (ECM) proteins in the adult OFC including WFA (55%), Brevican (32%), Neurocan (105%), Tenacin-C (25%), and HABP (5%). These findings are consistent with AIE causing persistent changes in brain structure that could contribute to a lack of behavioral flexibility.

  9. Adolescent binge ethanol treatment alters adult brain regional volumes, cortical extracellular matrix protein and behavioral flexibility

    PubMed Central

    Coleman, Leon Garland; Liu, Wen; Oguz, Ipek; Styner, Martin; Crews, Fulton T.

    2014-01-01

    Adolescents binge drink more than any other age group, increasing risk of disrupting the development of the frontal cortex. We hypothesized that adolescent binge drinking would lead to persistent alterations in adulthood. In this study, we modeled adolescent weekend underage binge-drinking, using adolescent mice (post-natal days [P] 28–37). The adolescent intermittent binge ethanol (AIE) treatment includes 6 binge intragastric doses of ethanol in an intermittent pattern across adolescence. Assessments were conducted in adulthood following extended abstinence to determine if there were persistent changes in adults. Reversal learning, open field and other behavioral assessments as well as brain structure using magnetic imaging and immunohistochemistry were determined. We found AIE did not impact adult Barnes Maze learning. However, AIE did cause reversal learning deficits in adults. AIE also caused structural changes in the adult brain. AIE was associated with adulthood volume enlargements in specific brain regions without changes in total brain volume. Enlarged regions included the orbitofrontal cortex (OFC, 4%), cerebellum (4.5%), thalamus (2%), internal capsule (10%) and genu of the corpus callosum (7%). The enlarged OFC volume in adults after AIE is consistent with previous imaging studies in human adolescents. AIE treatment was associated with significant increases in the expression of several extracellular matrix (ECM) proteins in the adult OFC including WFA (55%), Brevican (32%), Neurocan (105%), Tenacin-C (25%), and HABP (5%). These findings are consistent with AIE causing persistent changes in brain structure that could contribute to a lack of behavioral flexibility. PMID:24275185

  10. CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site.

    PubMed

    Kurcinski, Mateusz; Jamroz, Michal; Blaszczyk, Maciej; Kolinski, Andrzej; Kmiecik, Sebastian

    2015-07-01

    Protein-peptide interactions play a key role in cell functions. Their structural characterization, though challenging, is important for the discovery of new drugs. The CABS-dock web server provides an interface for modeling protein-peptide interactions using a highly efficient protocol for the flexible docking of peptides to proteins. While other docking algorithms require pre-defined localization of the binding site, CABS-dock does not require such knowledge. Given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), CABS-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone. This protocol was extensively tested over the largest dataset of non-redundant protein-peptide interactions available to date (including bound and unbound docking cases). For over 80% of bound and unbound dataset cases, we obtained models with high or medium accuracy (sufficient for practical applications). Additionally, as optional features, CABS-dock can exclude user-selected binding modes from docking search or to increase the level of flexibility for chosen receptor fragments. CABS-dock is freely available as a web server at http://biocomp.chem.uw.edu.pl/CABSdock.

  11. Identification of an Endocytosis Motif in an Intracellular Loop of Wntless Protein, Essential for Its Recycling and the Control of Wnt Protein Signaling*

    PubMed Central

    Gasnereau, Isabelle; Herr, Patrick; Chia, Pei Zhi Cheryl; Basler, Konrad; Gleeson, Paul A.

    2011-01-01

    The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of WlsAEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila WlsAEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins. PMID:22027831

  12. Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling.

    PubMed

    Gasnereau, Isabelle; Herr, Patrick; Chia, Pei Zhi Cheryl; Basler, Konrad; Gleeson, Paul A

    2011-12-16

    The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXϕ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls(AEGL) was rescued by the insertion of a classical YXXϕ motif in the cytoplasmic tail. Significantly, a Drosophila Wls(AEGL) mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXϕ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.

  13. U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

    PubMed Central

    Singh, Jagjit; Sikand, Kavleen; Conrad, Heike; Will, Cindy L.; Komar, Anton A.; Shukla, Girish C.

    2016-01-01

    Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome. PMID:27510544

  14. Activation loop phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length

    PubMed Central

    Cao, Muqing; Meng, Dan; Wang, Liang; Bei, Shuqing; Snell, William J.; Pan, Junmin

    2013-01-01

    Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift–associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift–associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly. PMID:23836633

  15. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2

    PubMed Central

    Kaur, Parminder; Wu, Dong; Lin, Jiangguo; Countryman, Preston; Bradford, Kira C.; Erie, Dorothy A.; Riehn, Robert; Opresko, Patricia L.; Wang, Hong

    2016-01-01

    Shelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways. PMID:26856421

  16. Identification of the Neutralizing Epitopes of Merkel Cell Polyomavirus Major Capsid Protein within the BC and EF Surface Loops

    PubMed Central

    Fleury, Maxime J. J.; Nicol, Jérôme T. J.; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  17. A Negative Feedback Loop between PHYTOCHROME INTERACTING FACTORs and HECATE Proteins Fine-Tunes Photomorphogenesis in Arabidopsis

    PubMed Central

    Zhu, Ling; Bu, Qingyun; Shen, Hui; Dang, Jonathan

    2016-01-01

    The phytochrome interacting factors (PIFs), a small group of basic helix-loop-helix transcription factors, repress photomorphogenesis both in the dark and light. Light signals perceived by the phytochrome family of photoreceptors induce rapid degradation of PIFs to promote photomorphogenesis. Here, we show that HECATE (HEC) proteins, another small group of HLH proteins, antagonistically regulate PIFs to promote photomorphogenesis. HEC1 and HEC2 heterodimerize with PIF family members. PIF1, HEC1, and HEC2 genes are spatially and temporally coexpressed, and HEC2 is localized in the nucleus. hec1, hec2, and hec3 single mutants and the hec1 hec2 double mutant showed hyposensitivity to light-induced seed germination and accumulation of chlorophyll and carotenoids, hallmark processes oppositely regulated by PIF1. HEC2 inhibits PIF1 target gene expression by directly heterodimerizing with PIF1 and preventing DNA binding and transcriptional activation activity of PIF1. Conversely, PIFs directly activate the expression of HEC1 and HEC2 in the dark, and light reduces the expression of these HECs possibly by degrading PIFs. HEC2 is partially degraded in the dark through the ubiquitin/26S-proteasome pathway and is stabilized by light. HEC2 overexpression also reduces the light-induced degradation of PIF1. Taken together, these data suggest that PIFs and HECs constitute a negative feedback loop to fine-tune photomorphogenesis in Arabidopsis thaliana. PMID:27073231

  18. A P-loop Mutation in G[alpha] Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

    SciTech Connect

    Bosch, Dustin E.; Willard, Francis S.; Ramanujam, Ravikrishna; Kimple, Adam J.; Willard, Melinda D.; Naqvi, Naweed I.; Siderovski, David P.

    2012-10-23

    Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active G{alpha}{beta}{gamma} heterotrimer relies on nucleotide cycling by the G{alpha} subunit: exchange of GTP for GDP activates G{alpha}, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting G{alpha} to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of G{alpha} subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that G{alpha}(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon G{alpha}{sub i1}(G42R) binding to GDP {center_dot} AlF{sub 4}{sup -} or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. G{alpha}(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with G{beta}{gamma} and GoLoco motifs in any nucleotide state. The corresponding G{alpha}{sub q}(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the G{alpha} subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two G{alpha} mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

  19. The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

    NASA Astrophysics Data System (ADS)

    Knight, Jonathan D.; Li, Rong; Botchan, Michael

    1991-04-01

    The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

  20. Seven Genes of the Enhancer of Split Complex of Drosophila Melanogaster Encode Helix-Loop-Helix Proteins

    PubMed Central

    Knust, E.; Schrons, H.; Grawe, F.; Campos-Ortega, J. A.

    1992-01-01

    Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells. PMID:1427040

  1. Structure of the flexible amino terminal domain of prion protein bound to a sulfated glycan

    PubMed Central

    Taubner, Lara M.; Bienkiewicz, Ewa A.; Copié, Valérie; Caughey, Byron

    2010-01-01

    The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence PHGGGWGQ that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear (1H, 13C, 15N) nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23–106 (PrP 23–106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23–106 remains disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data, which demonstrate that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan sidechains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23–106 when bound to PPS, consistent with the alignment of tryptophan sidechains. Chemical shift mapping identified binding sites on PrP 23–106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations. PMID:19913031

  2. Structure of the flexible amino-terminal domain of prion protein bound to a sulfated glycan.

    PubMed

    Taubner, Lara M; Bienkiewicz, Ewa A; Copié, Valérie; Caughey, Byron

    2010-01-22

    The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear ((1)H, (13)C, (15)N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The beta-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations. Published by Elsevier Ltd.

  3. Regulative Loops, Step Loops and Task Loops

    ERIC Educational Resources Information Center

    VanLehn, Kurt

    2016-01-01

    This commentary suggests a generalization of the conception of the behavior of tutoring systems, which the target article characterized as having an outer loop that was executed once per task and an inner loop that was executed once per step of the task. A more general conception sees these two loops as instances of regulative loops, which…

  4. Regulative Loops, Step Loops and Task Loops

    ERIC Educational Resources Information Center

    VanLehn, Kurt

    2016-01-01

    This commentary suggests a generalization of the conception of the behavior of tutoring systems, which the target article characterized as having an outer loop that was executed once per task and an inner loop that was executed once per step of the task. A more general conception sees these two loops as instances of regulative loops, which…

  5. Dynameomics: Data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction

    PubMed Central

    Rysavy, Steven J; Beck, David AC; Daggett, Valerie

    2014-01-01

    Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼25–75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. PMID:25142412

  6. Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

    PubMed Central

    Zeng, Kang; Bastos, Ricardo Nunes

    2010-01-01

    Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A–TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A–TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation. PMID:21187329

  7. LUTE (Local Unpruned Tuple Expansion): Accurate Continuously Flexible Protein Design with General Energy Functions and Rigid Rotamer-Like Efficiency.

    PubMed

    Hallen, Mark A; Jou, Jonathan D; Donald, Bruce R

    2016-09-28

    Most protein design algorithms search over discrete conformations and an energy function that is residue-pairwise, that is, a sum of terms that depend on the sequence and conformation of at most two residues. Although modeling of continuous flexibility and of non-residue-pairwise energies significantly increases the accuracy of protein design, previous methods to model these phenomena add a significant asymptotic cost to design calculations. We now remove this cost by modeling continuous flexibility and non-residue-pairwise energies in a form suitable for direct input to highly efficient, discrete combinatorial optimization algorithms such as DEE/A* or branch-width minimization. Our novel algorithm performs a local unpruned tuple expansion (LUTE), which can efficiently represent both continuous flexibility and general, possibly nonpairwise energy functions to an arbitrary level of accuracy using a discrete energy matrix. We show using 47 design calculation test cases that LUTE provides a dramatic speedup in both single-state and multistate continuously flexible designs.

  8. Evidence supporting the existence of a NUPR1-like family of helix-loop-helix chromatin proteins related to, yet distinct from, AT hook-containing HMG proteins.

    PubMed

    Urrutia, Raul; Velez, Gabriel; Lin, Marisa; Lomberk, Gwen; Neira, Jose Luis; Iovanna, Juan

    2014-08-01

    NUPR1, a small chromatin protein, plays a critical role in cancer development, progression, and resistance to therapy. Here, using a combination of structural bioinformatics and molecular modeling methods, we report several novel findings that enhance our understanding of the biochemical function of this protein. We find that NUPR1 has been conserved throughout evolution, and over time it has undergone duplications and transpositions to form other transcriptional regulators. Using threading, homology-based molecular modeling, molecular mechanics calculations, and molecular dynamics simulations, we generated structural models for four of these proteins: NUPR1a, NUPR1b, NUPR2, and the NUPR-like domain of GTF2-I. Comparative analyses of these models combined with extensive linear motif identification reveal that these four proteins, though similar in their propensities for folding, differ in size, surface changes, and sites amenable for posttranslational modification. Lastly, taking NUPR1a as the paradigm for this family, we built models of a NUPR-DNA complex. Additional structural comparisons revealed that NUPR1 defines a new family of small-groove-binding proteins that share structural features with, yet are distinct from, helix-loop-helix AT-hook-containing HMG proteins. These models and inferences should lead to a better understanding of the function of this group of chromatin proteins, which play a critical role in the development of human malignant diseases.

  9. Conformation and hydrogen ion titration of proteins: a continuum electrostatic model with conformational flexibility.

    PubMed

    You, T J; Bashford, D

    1995-11-01

    A new method for including local conformational flexibility in calculations of the hydrogen ion titration of proteins using macroscopic electrostatic models is presented. Intrinsic pKa values and electrostatic interactions between titrating sites are calculated from an ensemble of conformers in which the positions of titrating side chains are systematically varied. The method is applied to the Asp, Glu, and Tyr residues of hen lysozyme. The effects of different minimization and/or sampling protocols for both single-conformer and multi-conformer calculations are studied. For single-conformer calculations it is found that the results are sensitive to the choice of all-hydrogen versus polar-hydrogen-only atomic models and to the minimization protocol chosen. The best overall agreement of single-conformer calculations with experiment is obtained with an all-hydrogen model and either a two-step minimization process or minimization using a high dielectric constant. Multi-conformational calculations give significantly improved agreement with experiment, slightly smaller shifts between model compound pKa values and calculated intrinsic pKa values, and reduced sensitivity of the intrinsic pKa calculations to the initial details of the structure compared to single-conformer calculations. The extent of these improvements depends on the type of minimization used during the generation of conformers, with more extensive minimization giving greater improvements. The ordering of the titrations of the active-site residues, Glu-35 and Asp-52, is particularly sensitive to the minimization and sampling protocols used. The balance of strong site-site interactions in the active site suggests a need for including site-site conformational correlations.

  10. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    NASA Astrophysics Data System (ADS)

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-10-01

    The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  11. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein.

    PubMed

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A; Iyer, Pooja; Lane, Fiona M; Graham, James F; Goldmann, Wilfred; Pinheiro, Teresa J T; Gill, Andrew C

    2015-10-22

    The β2-α2 loop of PrP(C) is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrP(C) appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that 'rigidity' in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  12. Intrinsic Disorder in Pathogen Effectors: Protein Flexibility as an Evolutionary Hallmark in a Molecular Arms Race[W

    PubMed Central

    Marín, Macarena; Uversky, Vladimir N.; Ott, Thomas

    2013-01-01

    Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants’ innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein–protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors. PMID:24038649

  13. Docking ligands into flexible and solvated macromolecules. 4. Are popular scoring functions accurate for this class of proteins?

    PubMed

    Englebienne, Pablo; Moitessier, Nicolas

    2009-06-01

    In our previous report, we investigated the impact of protein flexibility and the presence of water molecules on the pose-prediction accuracy of major docking programs. To complete these investigations, we report herein a study of the impact of these two aspects on the accuracy of scoring functions. To this effect, we developed two sets of protein/ligand complexes made up of ligands cross-docked or cocrystallized with a large variety of proteins, featuring bridging water molecules and demonstrating protein flexibility. Efforts were made to reduce the correlation between the molecular weights of the selected ligands and their binding affinities, a major bias in some previously reported benchmark sets. Using these sets, 18 available scoring functions have been assessed for their accuracy to predict binding affinities and to rank-order compounds by their affinity to cocrystallized proteins. This study confirmed the good and similar accuracy of Xscore, GlideScore, DrugScore(CSD), GoldScore, PLP1, ChemScore, RankScore, and the eHiTS scoring function. Our next investigations demonstrated that most of the assessed scoring functions were much less accurate when the correct protein conformation was not provided. This study also revealed that considering the water molecules for scoring does not greatly affect the accuracy. Finally, this work sheds light on the high correlation between scoring functions and the poor increase in accuracy one can expect from consensus scoring.

  14. Structure of RNA Stem Loop B from the Picornavirus Replication Platform.

    PubMed

    Warden, Meghan S; Tonelli, Marco; Cornilescu, Gabriel; Liu, Dong; Hopersberger, Lorelei J; Ponniah, Komala; Pascal, Steven M

    2017-05-23

    The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy. SLB adopts a predominantly A-form helical structure. The stem contains five Watson-Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations into the helical parameters but does not appear to introduce flexibility. However, the helix major groove appears to be accessible. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the polyC-binding protein, is the most disordered region of the structure.

  15. Molecular characterization and expression of an oocyte-specific histone stem-loop binding protein in Carassius gibelio.

    PubMed

    Liu, Zhen; Zhang, Xiao-Juan; Wang, Wei; Zhang, Jun; Li, Zhi; Gui, Jian-Fang

    2015-12-01

    Stem-loop-binding proteins (SLBPs) have been revealed to interact with stem-loop of histones, and oocyte-specific and oocyte-preferential SLBP2 have been identified in vertebrates including Xenopus (S. tropicalis) and B. taurus to play a key role in histone translation regulation, but no oocyte-specific SLBPs have been characterized in fish. Here, we have identified and characterized the first fish oocyte-specific SLBP2 in Carassius gibelio. Its full-length cDNA contains 975 bp ORF encoding 324 amino acids. Firstly, the polyclonal antibody specific to C. gibelio SLBP2 was prepared. Then, RT-PCR analysis and Western blot detection revealed its oocyte-specific and dynamic expression pattern during oogenesis and embryogenesis of C. gibelio. Moreover, in situ hybridization and immunofluorescence localization observed its abundant expression in cortical alveolar stage oocytes and dynamic distribution in different stage oocytes. Altogether, our current data suggest that C. gibelio SLBP2 might play significant role in the early oogenesis and oocyte growth.

  16. 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.

  17. Crystal structure and mutagenesis of a protein phosphatase-1:calcineurin hybrid elucidate the role of the beta12-beta13 loop in inhibitor binding.

    PubMed

    Maynes, Jason T; Perreault, Kathleen R; Cherney, Maia M; Luu, Hue Anh; James, Michael N G; Holmes, Charles F B

    2004-10-08

    Protein phosphatase-1 and protein phosphatase-2B (calcineurin) are eukaryotic serine/threonine phosphatases that share 40% sequence identity in their catalytic subunits. Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by natural product toxins, such as microcystin-LR and okadaic acid. The most prominent region of non-conserved sequence between these phosphatases corresponds to the beta12-beta13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the beta12-beta13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) to the corresponding residues in calcineurin (312-316), resulted in a chimeric mutant that showed a decrease in sensitivity to microcystin-LR, okadaic acid, and the endogenous PP-1c inhibitor protein inhibitor-2. A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-A resolution. The beta12-beta13 loop region of the mutant superimposes closely with that of wild-type PP-1c bound to okadaic acid. Systematic mutation of each residue in the beta12-beta13 loop of PP-1c showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to toxins. Taken together, these data indicate that it is an individual amino acid residue substitution and not a change in the overall beta12-beta13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid.

  18. Specific Protein-Protein Interaction between Basic Helix-Loop-Helix Transcription Factors and Homeoproteins of the Pitx Family

    PubMed Central

    Poulin, Gino; Lebel, Mélanie; Chamberland, Michel; Paradis, Francois W.; Drouin, Jacques

    2000-01-01

    Homeoproteins and basic helix-loop-helix (bHLH) transcription factors are known for their critical role in development and cellular differentiation. The pituitary pro-opiomelanocortin (POMC) gene is a target for factors of both families. Indeed, pituitary-specific transcription of POMC depends on the action of the homeodomain-containing transcription factor Pitx1 and of bHLH heterodimers containing NeuroD1. We now show lineage-restricted expression of NeuroD1 in pituitary corticotroph cells and a direct physical interaction between bHLH heterodimers and Pitx1 that results in transcriptional synergism. The interaction between the bHLH and homeodomains is restricted to ubiquitous (class A) bHLH and to the Pitx subfamily. Since bHLH heterodimers interact with Pitx factors through their ubiquitous moiety, this mechanism may be implicated in other developmental processes involving bHLH factors, such as neurogenesis and myogenesis. PMID:10848608

  19. NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein.

    PubMed Central

    DeJong, Eric S; Marzluff, William F; Nikonowicz, Edward P

    2002-01-01

    The 3' end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U.A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5' flanking nucleotides are a critical component of the SLBP binding site. PMID:11871662

  20. The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane.

    PubMed

    Fontana, Juan; Atanasiu, Doina; Saw, Wan Ting; Gallagher, John R; Cox, Reagan G; Whitbeck, J Charles; Brown, Lauren M; Eisenberg, Roselyn J; Cohen, Gary H

    2017-08-22

    All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB's conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.IMPORTANCE The herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope

  1. Ensemble Generation and the Influence of Protein Flexibility on Geometric Tunnel Prediction in Cytochrome P450 Enzymes

    PubMed Central

    Kingsley, Laura J.; Lill, Markus A.

    2014-01-01

    Computational prediction of ligand entry and egress paths in proteins has become an emerging topic in computational biology and has proven useful in fields such as protein engineering and drug design. Geometric tunnel prediction programs, such as Caver3.0 and MolAxis, are computationally efficient methods to identify potential ligand entry and egress routes in proteins. Although many geometric tunnel programs are designed to accommodate a single input structure, the increasingly recognized importance of protein flexibility in tunnel formation and behavior has led to the more widespread use of protein ensembles in tunnel prediction. However, there has not yet been an attempt to directly investigate the influence of ensemble size and composition on geometric tunnel prediction. In this study, we compared tunnels found in a single crystal structure to ensembles of various sizes generated using different methods on both the apo and holo forms of cytochrome P450 enzymes CYP119, CYP2C9, and CYP3A4. Several protein structure clustering methods were tested in an attempt to generate smaller ensembles that were capable of reproducing the data from larger ensembles. Ultimately, we found that by including members from both the apo and holo data sets, we could produce ensembles containing less than 15 members that were comparable to apo or holo ensembles containing over 100 members. Furthermore, we found that, in the absence of either apo or holo crystal structure data, pseudo-apo or –holo ensembles (e.g. adding ligand to apo protein throughout MD simulations) could be used to resemble the structural ensembles of the corresponding apo and holo ensembles, respectively. Our findings not only further highlight the importance of including protein flexibility in geometric tunnel prediction, but also suggest that smaller ensembles can be as capable as larger ensembles at capturing many of the protein motions important for tunnel prediction at a lower computational cost. PMID

  2. High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

    PubMed Central

    Orita, M; Nishikawa, F; Kohno, T; Senda, T; Mitsui, Y; Yaeta, E; Kazunari, T; Nishikawa, S

    1996-01-01

    Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain. PMID:8604301

  3. Rationally designed coiled-coil DNA looping peptides control DNA topology

    PubMed Central

    Gowetski, Daniel B.; Kodis, Erin J.; Kahn, Jason D.

    2013-01-01

    Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein–DNA nanostructures. PMID:23825092

  4. A Feed Forward Loop Involving Protein Kinase C Alpha and MicroRNAs Regulates Tumor Cell Cycle

    PubMed Central

    Cohen, Ezra Eddy Wyssam; Zhu, Hongyan; Lingen, Mark W.; Martin, Leslie E.; Kuo, Wen-Liang; Choi, Eugene A.; Kocherginsky, Masha; Parker, Joel S.; Chung, Christine H.; Rosner, Marsha Rich

    2009-01-01

    Protein Kinase C alpha (PKCα) has been implicated in cancer but the mechanism is largely unknown. Here we show that PKCα promotes head and neck squamous cell carcinoma (SCCHN) by a feed forward network leading to cell cycle deregulation. PKCα inhibitors decrease proliferation in SCCHN cell lines and xenografted tumors. PKCα inhibition or depletion in tumor cells decreases DNA synthesis by suppressing ERK phosphorylation and cyclin E synthesis. Additionally, PKCα down-regulates miR-15a, a microRNA that directly inhibits protein synthesis of cyclin E as well as other cell cycle regulators. Furthermore, both PKCα and cyclin E protein expression are increased in primary tumors, and PKCα inversely correlates with miR15a expression in primary tumors. Finally, PKCα is associated with poor prognosis in SCCHN. These results identify PKCα as a key regulator of HNSCC tumor cell growth by a mechanism involving activation of MAP kinase, an initiator of the cell cycle, and suppression of miR-15a, an inhibitor of DNA synthesis. Although the specific components may be different, this type of feed forward loop network, consisting of a stimulus that activates a positive signal and removes a negative brake, is likely to be a general one that enables induction of DNA synthesis by a variety of growth or oncogenic stimuli. PMID:19117988

  5. Mutations on the FG surface loop of human papillomavirus type 16 major capsid protein affect recognition by both type-specific neutralizing antibodies and cross-reactive antibodies.

    PubMed

    Carpentier, Guillaume S; Fleury, Maxime J J; Touzé, Antoine; Sadeyen, Jean-Rémy; Tourne, Sylvie; Sizaret, Pierre-Yves; Coursaget, Pierre

    2005-12-01

    The aim of this study was to further characterize the conformational neutralizing epitopes present on the surface-exposed FG loop of human papillomavirus (HPV) type 16 L1 major capsid protein. We have generated previously two chimeric L1 proteins by insertion of a foreign peptide encoding an epitope of the hepatitis B core (HBc) antigen within the FG loop. In addition, three other chimeric L1 proteins were obtained by replacing three different FG loop sequences by the HBc motif and three others by point mutations. All these chimeric L1 proteins retained the ability to self-assemble into virus-like particles (VLPs), with the exception of the mutant with substitution of the L1 sequence 274-279 by the HBc motif. The eight chimeric VLPs were then analyzed for differential reactivity with a set of six HPV-16 and HPV-31 monoclonal antibodies that bound to conformational and linear epitopes. The binding patterns of these monoclonal antibodies confirmed that the FG loop contained or contributed to neutralizing conformational epitopes. The results obtained suggested that the H31.F7 antibody, an anti-HPV-31 cross-reacting and neutralizing antibody, recognized a conformational epitope situated before the 266-271 sequence. In addition, H16.E70 neutralizing antibody reactivity was reduced with L1 VLPs with an Asn to Ala point mutation at position 270, suggesting that Asn is a part of the epitope recognized by this antibody. This study contributes to the understanding of the antigenic structure of HPV-16 and -31 L1 proteins by confirming that the FG loop contributes to neutralizing epitopes and suggesting the existence of both type-specific and cross-reactive conformational epitopes within the FG loop. Copyright (c) 2005 Wiley-Liss, inc.

  6. The Energy Landscape of Hyperstable LacI-DNA Loops

    NASA Astrophysics Data System (ADS)

    Kahn, Jason

    2009-03-01

    The Escherichia coli LacI protein represses transcription of the lac operon by blocking access to the promoter through binding at a promoter-proximal DNA operator. The affinity of tetrameric LacI (and therefore the repression efficiency) is enhanced by simultaneous binding to an auxiliary operator, forming a DNA loop. Hyperstable LacI-DNA loops were previously shown to be formed on DNA constructs that include a sequence-directed bend flanked by operators. Biochemical experiments showed that two such constructs (9C14 and 11C12) with different helical phasing between the operators and the DNA bend form different DNA loop shapes. The geometry and topology of the loops and the relevance of alternative conformations suggested by probable flexible linkers in LacI remain unclear. Bulk and single molecule fluorescence resonance energy transfer (SM-FRET, with D. English) experiments on a dual fluorophore-labeled 9C14-LacI loop demonstrate that it adopts a single, stable, rigid closed-form loop conformation. Here, we characterize the LacI-9C14 loop by SM-FRET as a function of inducer isopropyl-β,D-thiogalactoside (IPTG) concentration. Energy transfer measurements reveal partial but incomplete destabilization of loop formation by IPTG. Surprisingly, there is no change in the energy transfer efficiency of the remaining looped population. Models for the regulation of the lac operon often assume complete disruption of LacI-operator complexes upon inducer binding to LacI. Our work shows that even at saturating IPTG there is still a significant population of LacI-DNA complexes in a looped state, in accord with previous in vivo experiments that show incomplete induction (with J. Maher). Finally, we will report progress on characterizing the ``energy landscape'' for DNA looping upon systematic variation of the DNA linkers between the operators and the bending locus. Rod mechanics simulations (with N. Perkins) provide testable predictions on loop stability, topology, and FRET.

  7. Myogenic basic helix-loop-helix proteins regulate the expression of peroxisomal proliferator activated receptor-gamma coactivator-1alpha.

    PubMed

    Chang, Ju Hui; Lin, Kwang Huei; Shih, Chung Hsuan; Chang, Yu Jung; Chi, Hsiang Chung; Chen, Shen Liang

    2006-06-01

    Peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of the PGC-1alpha gene in either a cell or an animal has been shown to promote fast to slow fiber-type switch. The expression of PGC-1alpha in muscle is regulated by myocyte enhancer factor 2 and Forkhead in rhabdomyosarcoma, two transcription factors implicated in terminal muscle differentiation. In this study we found that PGC-1alpha expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus-mediated MyoD overexpression in C3H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1alpha expression in vivo. Our transient transfection results also show that myogenic basic helix-loop-helix (bHLH) proteins, especially MyoD, can activate PGC-1alpha expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1alpha promoter were localized to a short region (-49 to approximately +2) adjacent to the transcription start site, which contains two putative E boxes. Mutation of either site significantly reduced MyoD-mediated transactivation in the cells, suggesting that both sites are required for myogenic bHLH protein-mediated activation. However, only one site, the E2 box, was directly bound by glutathione-S-transferase-MyoD protein in EMSAs. Our results indicate that myogenic bHLH proteins not only are involved in lineage determination and terminal differentiation, but also are directly implicated in activation of the key fiber-type and metabolic switch gene, PGC-1alpha.

  8. Benzimidazole inhibitors of the protein kinase CHK2: clarification of the binding mode by flexible side chain docking and protein-ligand crystallography.

    PubMed

    Matijssen, Cornelis; Silva-Santisteban, M Cris; Westwood, Isaac M; Siddique, Samerene; Choi, Vanessa; Sheldrake, Peter; van Montfort, Rob L M; Blagg, Julian

    2012-11-15

    Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.

  9. Chain Flexibility in Self-Assembled Monolayers Affects Protein Adsorption and Surface Hydration: A Molecular Dynamics Study.

    PubMed

    Beckner, Wesley; He, Yi; Pfaendtner, Jim

    2016-10-04

    Recent developments in the antifouling properties of Self-Assembled Monolayers (SAMs) have largely focused on increasing the enthalpic association of a hydration layer along the interface of those surfaces with water. However, an entropic penalty due to chain restriction also disfavors biomolecule-surface adsorption. To isolate the effect of this entropic penalty amid changing packing densities, molecular dynamics simulations of explicitly solvated systems of lysozyme and seven monomer length oligo (ethylene glycol) (OEG) SAMs were performed. SAM surfaces were constructed at 100%, 74%, and 53% of a maximum packing (MP) density of 4.97 Å interchain spacing and the effect of chain flexibility was isolated by selectively freezing chain monomers. The rate of protein adsorption as well as the conformation and orientation of the protein upon adsorption were examined. It was found that chain spacing was a strong determinant in adsorption properties while chain flexibility played a secondary role. Of the three packing densities, 74% of MP was the most antifouling with increased antifouling behavior at moderate chain flexibility, i.e. two to four free monomer groups.

  10. CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site

    PubMed Central

    Kurcinski, Mateusz; Jamroz, Michal; Blaszczyk, Maciej; Kolinski, Andrzej; Kmiecik, Sebastian

    2015-01-01

    Protein–peptide interactions play a key role in cell functions. Their structural characterization, though challenging, is important for the discovery of new drugs. The CABS-dock web server provides an interface for modeling protein–peptide interactions using a highly efficient protocol for the flexible docking of peptides to proteins. While other docking algorithms require pre-defined localization of the binding site, CABS-dock does not require such knowledge. Given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), CABS-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone. This protocol was extensively tested over the largest dataset of non-redundant protein–peptide interactions available to date (including bound and unbound docking cases). For over 80% of bound and unbound dataset cases, we obtained models with high or medium accuracy (sufficient for practical applications). Additionally, as optional features, CABS-dock can exclude user-selected binding modes from docking search or to increase the level of flexibility for chosen receptor fragments. CABS-dock is freely available as a web server at http://biocomp.chem.uw.edu.pl/CABSdock. PMID:25943545

  11. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

    PubMed Central

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-01-01

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations. PMID:26879383

  12. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity.

    PubMed

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-02-16

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 3(10)-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 3(10)-helix and helps to stabilize the active conformation. The coil-3(10)-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

  13. Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor

    PubMed Central

    Parker, Lorien J.; Watanabe, Hisami; Tsuganezawa, Keiko; Tomabechi, Yuri; Handa, Noriko; Shirouzu, Mikako; Yuki, Hitomi; Honma, Teruki; Ogawa, Naoko; Nagano, Tetsuo; Yokoyama, Shigeyuki; Tanaka, Akiko

    2012-01-01

    The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012 ▶), J. Med. Chem. 55, 5151–5156]. The report described the process of optimization of the structure–activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-­yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the β3 strand located above the binding site, is not usually observed in Pim-1 structures. PMID:22869110

  14. Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor.

    PubMed

    Parker, Lorien J; Watanabe, Hisami; Tsuganezawa, Keiko; Tomabechi, Yuri; Handa, Noriko; Shirouzu, Mikako; Yuki, Hitomi; Honma, Teruki; Ogawa, Naoko; Nagano, Tetsuo; Yokoyama, Shigeyuki; Tanaka, Akiko

    2012-08-01

    The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the β3 strand located above the binding site, is not usually observed in Pim-1 structures.

  15. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-02-01

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

  16. Structural flexibility of the periplasmic protein, FlgA, regulates flagellar P-ring assembly in Salmonella enterica

    PubMed Central

    Matsunami, Hideyuki; Yoon, Young-Ho; Meshcheryakov, Vladimir A.; Namba, Keiichi; Samatey, Fadel A.

    2016-01-01

    A periplasmic flagellar chaperone protein, FlgA, is required for P-ring assembly in bacterial flagella of taxa such as Salmonella enterica or Escherichia coli. The mechanism of chaperone-mediated P-ring formation is poorly understood. Here we present the open and closed crystal structures of FlgA from Salmonella enterica serovar Typhimurium, grown under different crystallization conditions. An intramolecular disulfide cross-linked form of FlgA caused a dominant negative effect on motility of the wild-type strain. Pull-down experiments support a specific protein-protein interaction between FlgI, the P-ring component protein, and the C-terminal domain of FlgA. Surface plasmon resonance and limited-proteolysis indicate that flexibility of the domain is reduced in the covalently closed form. These results show that the structural flexibility of the C-terminal domain of FlgA, which is related to the structural difference between the two crystal forms, is intrinsically associated with its molecular chaperone function in P-ring assembly. PMID:27273476

  17. Modeling of protein-peptide interactions using the CABS-dock web server for binding site search and flexible docking.

    PubMed

    Blaszczyk, Maciej; Kurcinski, Mateusz; Kouza, Maksim; Wieteska, Lukasz; Debinski, Aleksander; Kolinski, Andrzej; Kmiecik, Sebastian

    2016-01-15

    Protein-peptide interactions play essential functional roles in living organisms and their structural characterization is a hot subject of current experimental and theoretical research. Computational modeling of the structure of protein-peptide interactions is usually divided into two stages: prediction of the binding site at a protein receptor surface, and then docking (and modeling) the peptide structure into the known binding site. This paper presents a comprehensive CABS-dock method for the simultaneous search of binding sites and flexible protein-peptide docking, available as a user's friendly web server. We present example CABS-dock results obtained in the default CABS-dock mode and using its advanced options that enable the user to increase the range of flexibility for chosen receptor fragments or to exclude user-selected binding modes from docking search. Furthermore, we demonstrate a strategy to improve CABS-dock performance by assessing the quality of models with classical molecular dynamics. Finally, we discuss the promising extensions and applications of the CABS-dock method and provide a tutorial appendix for the convenient analysis and visualization of CABS-dock results. The CABS-dock web server is freely available at http://biocomp.chem.uw.edu.pl/CABSdock/.

  18. Multi-Conformation Monte Carlo: A Method for Introducing Flexibility in Efficient Simulations of Many-Protein Systems.

    PubMed

    Prytkova, Vera; Heyden, Matthias; Khago, Domarin; Freites, J Alfredo; Butts, Carter T; Martin, Rachel W; Tobias, Douglas J

    2016-08-25

    We present a novel multi-conformation Monte Carlo simulation method that enables the modeling of protein-protein interactions and aggregation in crowded protein solutions. This approach is relevant to a molecular-scale description of realistic biological environments, including the cytoplasm and the extracellular matrix, which are characterized by high concentrations of biomolecular solutes (e.g., 300-400 mg/mL for proteins and nucleic acids in the cytoplasm of Escherichia coli). Simulation of such environments necessitates the inclusion of a large number of protein molecules. Therefore, computationally inexpensive methods, such as rigid-body Brownian dynamics (BD) or Monte Carlo simulations, can be particularly useful. However, as we demonstrate herein, the rigid-body representation typically employed in simulations of many-protein systems gives rise to certain artifacts in protein-protein interactions. Our approach allows us to incorporate molecular flexibility in Monte Carlo simulations at low computational cost, thereby eliminating ambiguities arising from structure selection in rigid-body simulations. We benchmark and validate the methodology using simulations of hen egg white lysozyme in solution, a well-studied system for which extensive experimental data, including osmotic second virial coefficients, small-angle scattering structure factors, and multiple structures determined by X-ray and neutron crystallography and solution NMR, as well as rigid-body BD simulation results, are available for comparison.

  19. Automated Docking with Protein Flexibility in the Design of Femtomolar “Click Chemistry” Inhibitors of Acetylcholinesterase

    PubMed Central

    Morris, Garrett M.; Green, Luke G.; Radić, Zoran; Taylor, Palmer; Sharpless, K. Barry; Olson, Arthur J.; Grynszpan, Flavio

    2013-01-01

    The use of computer-aided structure-based drug design prior to synthesis has proven to be generally valuable in suggesting improved binding analogues of existing ligands.1 Here we describe the application of the program AutoDock2 to the design of a focused library that was used in the “click chemistry in-situ” generation of the most potent non-covalent inhibitor of the enzyme acetylcholinesterase (AChE) yet developed (Kd = ~100 fM).3 AutoDock version 3.0.5 has been widely distributed and successfully used to predict bound conformations of flexible ligands. Here, we also used a version of AutoDock which permits additional conformational flexibility in selected amino acid sidechains of the target protein. PMID:23451944

  20. Dead-End Elimination with Perturbations (“DEEPer”): A provable protein design algorithm with continuous sidechain and backbone flexibility

    PubMed Central

    Hallen, Mark A.; Keedy, Daniel A.; Donald, Bruce R.

    2012-01-01

    Computational protein and drug design generally require accurate modeling of protein conformations. This modeling typically starts with an experimentally-determined protein structure and considers possible conformational changes due to mutations or new ligands. The DEE/A* algorithm provably finds the GMEC (global minimum-energy conformation) of a protein assuming the backbone does not move and the sidechains take on conformations from a set of discrete, experimentally-observed conformations called rotamers. DEE/A* can efficiently find the overall GMEC for exponentially many mutant sequences. Previous improvements to DEE/A* include modeling ensembles of sidechain conformations and either continuous sidechain or backbone flexibility. We present a new algorithm, DEEPer (Dead-End Elimination with Perturbations), that combines these advantages and can also handle much more extensive backbone flexibility and backbone ensembles. DEEPer provably finds the GMEC or, if desired by the user, all conformations and sequences within a specified energy window of the GMEC. It includes the new abilities to handle arbitrarily large backbone perturbations and to generate ensembles of backbone conformations. It also incorporates the shear, an experimentally-observed local backbone motion never before used in design. Additionally, we derive a new method to accelerate DEE/A*-based calculations, indirect pruning, that is particularly useful for DEEPer. In 67 benchmark tests on 64 proteins, DEEPer consistently identified lower-energy conformations than previous methods did, indicating more accurate modeling. Additional tests demonstrated its ability to incorporate larger, experimentally-observed backbone conformational changes and to model realistic conformational ensembles. These capabilities provide significant advantages for modeling protein mutations and protein-ligand interactions. PMID:22821798

  1. Loop quantization

    SciTech Connect

    Nicolau, A.

    1988-10-01

    Loop unwinding is a known technique for reducing loop overhead, exposing parallelism, and increasing the efficiency of pipelining. Traditional loop unwinding is limited to the innermost loop in a group of nested loops and the amount of unwinding either is fixed or must be specified by the user, on a case by case basis. In this paper the authors present a general technique for automatically unwinding multiply nested loops, explain its advantages over other transformation techniques, and illustrate its practical effectiveness. Lopp Quantization could be beneficial by itself or coupled with other loop transformations.

  2. DNA Flexibility

    NASA Astrophysics Data System (ADS)

    Widom, Jonathan

    2005-03-01

    Classic experimental and theoretical analyses led to a unified view of DNA as a semiflexible polymer, characterized by a bending persistence length, P, ˜50 nm (˜150 bp). In this view, DNA lengths that are greater than P are, on average, spontaneously gently bent, and require relatively little force to bend significantly, while DNA lengths that are shorter than P are nearly straight, and require great force to bend significantly. Nevertheless, sharply bent DNA plays important roles in biology. We used the method of ligase catalyzed DNA cyclization to investigate the spontaneous looping of short DNAs. Remarkably, DNAs as short as 84 bp spontaneously bend into circles, and 94 bp DNA sequences cyclize up to 10^5 times more easily than predicted from classic theories of DNA bending. In subsequent studies we find that the twistability of sharply looped DNAs exceeds the prediction of classic theories by up to 400-fold. These results can only be understood by greatly enhanced DNA flexibility, not by permanent bends. Our results provide striking support for two new theories of DNA mechanics based on local melted or kinked regions, and they establish DNA as an active participant in the formation and function of looped regulatory complexes in vivo.

  3. Importance of chirality and reduced flexibility of protein side chains: A study with square and tetrahedral lattice models

    NASA Astrophysics Data System (ADS)

    Zhang, Jinfeng; Chen, Yu; Chen, Rong; Liang, Jie

    2004-07-01

    Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our

  4. Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes.

    PubMed

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

    2010-10-25

    The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGΔ) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGΔ in Vero cells at 37 °C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. Engineering covalent loops in proteins can serve as an on/off switch to regulate threaded topologies.

    PubMed

    Haglund, Ellinor

    2015-09-09

    Knots in proteins are under active investigation motivating refinements of current techniques and the development of tools to better understand the knotted topology. A strong focus is to identify new knots and expand upon the current understanding of their complex topology. Previous work has shown that the knotted topology, even in the simplest case of knots, encompasses a variety of unique challenges in folding and tying a chain. To bypass many of the in vitro experimental complications involved in working with knots, it is useful to apply methodologies to a more simplified system. The pierced lasso bundles (PLB), we discovered where a single disulphide bridge holds the threaded topology together, presents a simpler system to study knots in vitro. Having a disulphide bridge as an on/off switch between the threaded/unthreaded topology is advantageous because a covalent loop allows manipulation of the knot without directly altering affecting secondary and tertiary structure. Because disulphide bridges are commonly used in protein engineering, a pierced lasso (PL) topology can be easily introduced into a protein of interest to form a knotted topology within a given secondary structure. It is also important to take into account that if formed, disulphides can inadvertently introduce an unwanted PL. This was found upon determination of the crystal structure (PDB code 2YHG) of the recently de novo designed nucleoside hydrolase. Our detailed investigations of the PL presented here will allow researchers to look at the introduction of disulphide bridges in a larger context with respect to potential geometrical consequences on the structure and functional properties of proteins.

  6. N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

    PubMed

    Soto, Antonio G; Smith, Thomas H; Chen, Buxin; Bhattacharya, Supriyo; Cordova, Isabel Canto; Kenakin, Terry; Vaidehi, Nagarajan; Trejo, JoAnn

    2015-07-07

    Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

  7. Engineering covalent loops in proteins can serve as an on/off switch to regulate threaded topologies

    NASA Astrophysics Data System (ADS)

    Haglund, Ellinor

    2015-09-01

    Knots in proteins are under active investigation motivating refinements of current techniques and the development of tools to better understand the knotted topology. A strong focus is to identify new knots and expand upon the current understanding of their complex topology. Previous work has shown that the knotted topology, even in the simplest case of knots, encompasses a variety of unique challenges in folding and tying a chain. To bypass many of the in vitro experimental complications involved in working with knots, it is useful to apply methodologies to a more simplified system. The pierced lasso bundles (PLB), we discovered where a single disulphide bridge holds the threaded topology together, presents a simpler system to study knots in vitro. Having a disulphide bridge as an on/off switch between the threaded/unthreaded topology is advantageous because a covalent loop allows manipulation of the knot without directly altering affecting secondary and tertiary structure. Because disulphide bridges are commonly used in protein engineering, a pierced lasso (PL) topology can be easily introduced into a protein of interest to form a knotted topology within a given secondary structure. It is also important to take into account that if formed, disulphides can inadvertently introduce an unwanted PL. This was found upon determination of the crystal structure (PDB code 2YHG) of the recently de novo designed nucleoside hydrolase. Our detailed investigations of the PL presented here will allow researchers to look at the introduction of disulphide bridges in a larger context with respect to potential geometrical consequences on the structure and functional properties of proteins.

  8. Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice.

    PubMed

    Sweeney, Megan T; Thomson, Michael J; Pfeil, Bernard E; McCouch, Susan

    2006-02-01

    Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

  9. Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

    PubMed

    Sajman, Julia; Zenvirth, Drora; Nitzan, Mor; Margalit, Hanah; Simpson-Lavy, Kobi J; Reiss, Yuval; Cohen, Itamar; Ravid, Tommer; Brandeis, Michael

    2015-05-11

    Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner—both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.

  10. The β5-Loop and Lid Domain Contribute to the Substrate Specificity of Pancreatic Lipase-related Protein 2 (PNLIPRP2)*

    PubMed Central

    Xiao, Xunjun; Lowe, Mark E.

    2015-01-01

    Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2. PMID:26494624

  11. The β5-Loop and Lid Domain Contribute to the Substrate Specificity of Pancreatic Lipase-related Protein 2 (PNLIPRP2).

    PubMed

    Xiao, Xunjun; Lowe, Mark E

    2015-11-27

    Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2.

  12. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

    PubMed Central

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S.; Olson, Arthur J.; Sanner, Michel F.

    2015-01-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on t