SIMPLE estimate of the free energy change due to aliphatic mutations: superior predictions based on first principles.

PubMed

Bueno, Marta; Camacho, Carlos J; Sancho, Javier

2007-09-01

The bioinformatics revolution of the last decade has been instrumental in the development of empirical potentials to quantitatively estimate protein interactions for modeling and design. Although computationally efficient, these potentials hide most of the relevant thermodynamics in 5-to-40 parameters that are fitted against a large experimental database. Here, we revisit this longstanding problem and show that a careful consideration of the change in hydrophobicity, electrostatics, and configurational entropy between the folded and unfolded state of aliphatic point mutations predicts 20-30% less false positives and yields more accurate predictions than any published empirical energy function. This significant improvement is achieved with essentially no free parameters, validating past theoretical and experimental efforts to understand the thermodynamics of protein folding. Our first principle analysis strongly suggests that both the solute-solute van der Waals interactions in the folded state and the electrostatics free energy change of exposed aliphatic mutations are almost completely compensated by similar interactions operating in the unfolded ensemble. Not surprisingly, the problem of properly accounting for the solvent contribution to the free energy of polar and charged group mutations, as well as of mutations that disrupt the protein backbone remains open. 2007 Wiley-Liss, Inc.

Systematic approach to developing empirical interatomic potentials for III-N semiconductors

NASA Astrophysics Data System (ADS)

Ito, Tomonori; Akiyama, Toru; Nakamura, Kohji

2016-05-01

A systematic approach to the derivation of empirical interatomic potentials is developed for III-N semiconductors with the aid of ab initio calculations. The parameter values of empirical potential based on bond order potential are determined by reproducing the cohesive energy differences among 3-fold coordinated hexagonal, 4-fold coordinated zinc blende, wurtzite, and 6-fold coordinated rocksalt structures in BN, AlN, GaN, and InN. The bond order p is successfully introduced as a function of the coordination number Z in the form of p = a exp(-bZn ) if Z ≤ 4 and p = (4/Z)α if Z ≥ 4 in empirical interatomic potential. Moreover, the energy difference between wurtzite and zinc blende structures can be successfully evaluated by considering interaction beyond the second-nearest neighbors as a function of ionicity. This approach is feasible for developing empirical interatomic potentials applicable to a system consisting of poorly coordinated atoms at surfaces and interfaces including nanostructures.

The Intrinsically Disordered Regions of the Drosophila melanogaster Hox Protein Ultrabithorax Select Interacting Proteins Based on Partner Topology

PubMed Central

Hsiao, Hao-Ching; Gonzalez, Kim L.; Catanese, Daniel J.; Jordy, Kristopher E.; Matthews, Kathleen S.; Bondos, Sarah E.

2014-01-01

Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, we evaluated interactions formed by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and α-α superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues. PMID:25286318

Pairwise contact energy statistical potentials can help to find probability of point mutations.

PubMed

Saravanan, K M; Suvaithenamudhan, S; Parthasarathy, S; Selvaraj, S

2017-01-01

To adopt a particular fold, a protein requires several interactions between its amino acid residues. The energetic contribution of these residue-residue interactions can be approximated by extracting statistical potentials from known high resolution structures. Several methods based on statistical potentials extracted from unrelated proteins are found to make a better prediction of probability of point mutations. We postulate that the statistical potentials extracted from known structures of similar folds with varying sequence identity can be a powerful tool to examine probability of point mutation. By keeping this in mind, we have derived pairwise residue and atomic contact energy potentials for the different functional families that adopt the (α/β) 8 TIM-Barrel fold. We carried out computational point mutations at various conserved residue positions in yeast Triose phosphate isomerase enzyme for which experimental results are already reported. We have also performed molecular dynamics simulations on a subset of point mutants to make a comparative study. The difference in pairwise residue and atomic contact energy of wildtype and various point mutations reveals probability of mutations at a particular position. Interestingly, we found that our computational prediction agrees with the experimental studies of Silverman et al. (Proc Natl Acad Sci 2001;98:3092-3097) and perform better prediction than i Mutant and Cologne University Protein Stability Analysis Tool. The present work thus suggests deriving pairwise contact energy potentials and molecular dynamics simulations of functionally important folds could help us to predict probability of point mutations which may ultimately reduce the time and cost of mutation experiments. Proteins 2016; 85:54-64. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Computer Folding of RNA Tetraloops: Identification of Key Force Field Deficiencies.

PubMed

Kührová, Petra; Best, Robert B; Bottaro, Sandro; Bussi, Giovanni; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

2016-09-13

The computer-aided folding of biomolecules, particularly RNAs, is one of the most difficult challenges in computational structural biology. RNA tetraloops are fundamental RNA motifs playing key roles in RNA folding and RNA-RNA and RNA-protein interactions. Although state-of-the-art Molecular Dynamics (MD) force fields correctly describe the native state of these tetraloops as a stable free-energy basin on the microsecond time scale, enhanced sampling techniques reveal that the native state is not the global free energy minimum, suggesting yet unidentified significant imbalances in the force fields. Here, we tested our ability to fold the RNA tetraloops in various force fields and simulation settings. We employed three different enhanced sampling techniques, namely, temperature replica exchange MD (T-REMD), replica exchange with solute tempering (REST2), and well-tempered metadynamics (WT-MetaD). We aimed to separate problems caused by limited sampling from those due to force-field inaccuracies. We found that none of the contemporary force fields is able to correctly describe folding of the 5'-GAGA-3' tetraloop over a range of simulation conditions. We thus aimed to identify which terms of the force field are responsible for this poor description of TL folding. We showed that at least two different imbalances contribute to this behavior, namely, overstabilization of base-phosphate and/or sugar-phosphate interactions and underestimated stability of the hydrogen bonding interaction in base pairing. The first artifact stabilizes the unfolded ensemble, while the second one destabilizes the folded state. The former problem might be partially alleviated by reparametrization of the van der Waals parameters of the phosphate oxygens suggested by Case et al., while in order to overcome the latter effect we suggest local potentials to better capture hydrogen bonding interactions.

Systematics of α-decay fine structure in odd-mass nuclei based on a finite-range nucleon-nucleon interaction

NASA Astrophysics Data System (ADS)

Adel, A.; Alharbi, T.

2018-07-01

A systematic study on α-decay fine structure is presented for odd-mass nuclei in the range 83 ≤ Z ≤ 92. The α-decay partial half-lives and branching ratios to the ground and excited states of daughter nuclei are calculated in the framework of the Wentzel-Kramers-Brillouin (WKB) approximation with the implementation of the Bohr-Sommerfeld quantization condition. The microscopic α-daughter potential is obtained using the double-folding model with a realistic M3Y-Paris nucleon-nucleon (NN) interaction. The exchange potential, which accounts for the knock-on exchange of nucleons between the interacting nuclei, is calculated using the finite-range exchange NN interaction which is essentially a much better approximation as compared to the zero-range pseudo-potential adopted in the usual double-folding calculations. Our calculations of α-decay fine structure have been improved by considering the preformation factor extracted from the recently proposed cluster formation model on basis of the binding energy difference. The computed partial half-lives and branching ratios are compared with the recent experimental data and they are in good agreement.

Protein folding on Biosensor tips: Folding of Maltodextrin glucosidase monitored by its interactions with GroEL

PubMed Central

Pastor, Ashutosh; Singh, Amit K.; Fisher, Mark T.; Chaudhuri, Tapan K.

2016-01-01

Protein folding has been extensively studied for past four decades by employing solution based experiments such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR and HD exchange. However, for rapid analysis of the folding process, solution based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work we demonstrate that a Bio-Layer Interferometry (BLI) chaperonin detection system can be potentially applied to identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on Bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation prone protein Maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the Bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution. PMID:27367928

Semiempirical prediction of protein folds

NASA Astrophysics Data System (ADS)

Fernández, Ariel; Colubri, Andrés; Appignanesi, Gustavo

2001-08-01

We introduce a semiempirical approach to predict ab initio expeditious pathways and native backbone geometries of proteins that fold under in vitro renaturation conditions. The algorithm is engineered to incorporate a discrete codification of local steric hindrances that constrain the movements of the peptide backbone throughout the folding process. Thus, the torsional state of the chain is assumed to be conditioned by the fact that hopping from one basin of attraction to another in the Ramachandran map (local potential energy surface) of each residue is energetically more costly than the search for a specific (Φ, Ψ) torsional state within a single basin. A combinatorial procedure is introduced to evaluate coarsely defined torsional states of the chain defined ``modulo basins'' and translate them into meaningful patterns of long range interactions. Thus, an algorithm for structure prediction is designed based on the fact that local contributions to the potential energy may be subsumed into time-evolving conformational constraints defining sets of restricted backbone geometries whereupon the patterns of nonbonded interactions are constructed. The predictive power of the algorithm is assessed by (a) computing ab initio folding pathways for mammalian ubiquitin that ultimately yield a stable structural pattern reproducing all of its native features, (b) determining the nucleating event that triggers the hydrophobic collapse of the chain, and (c) comparing coarse predictions of the stable folds of moderately large proteins (N~100) with structural information extracted from the protein data bank.

Physiologically Based Pharmacokinetic Modeling Suggests Limited Drug–Drug Interaction Between Clopidogrel and Dasabuvir

PubMed Central

Fu, W; Badri, P; Bow, DAJ; Fischer, V

2017-01-01

Dasabuvir, a nonnucleoside NS5B polymerase inhibitor, is a sensitive substrate of cytochrome P450 (CYP) 2C8 with a potential for drug–drug interaction (DDI) with clopidogrel. A physiologically based pharmacokinetic (PBPK) model was developed for dasabuvir to evaluate the DDI potential with clopidogrel, the acyl‐β‐D glucuronide metabolite of which has been reported as a strong mechanism‐based inhibitor of CYP2C8 based on an interaction with repaglinide. In addition, the PBPK model for clopidogrel and its metabolite were updated with additional in vitro data. Sensitivity analyses using these PBPK models suggested that CYP2C8 inhibition by clopidogrel acyl‐β‐D glucuronide may not be as potent as previously suggested. The dasabuvir and updated clopidogrel PBPK models predict a moderate increase of 1.5–1.9‐fold for Cmax and 1.9–2.8‐fold for AUC of dasabuvir when coadministered with clopidogrel. While the PBPK results suggest there is a potential for DDI between dasabuvir and clopidogrel, the magnitude is not expected to be clinically relevant. PMID:28411400

Folding model analyses of 12C-12C and 16O-16O elastic scattering using the density-dependent LOCV-averaged effective interaction

NASA Astrophysics Data System (ADS)

Rahmat, M.; Modarres, M.

2018-03-01

The averaged effective two-body interaction (AEI), which can be generated through the lowest order constrained variational (LOCV) method for symmetric nuclear matter (SNM) with the input [Reid68, Ann. Phys. 50, 411 (1968), 10.1016/0003-4916(68)90126-7] nucleon-nucleon potential, is used as the effective nucleon-nucleon potential in the folding model to describe the heavy-ion (HI) elastic scattering cross sections. The elastic scattering cross sections of 12C-12C and 16O-16O systems are calculated in the above framework. The results are compared with the corresponding calculations coming from the fitting procedures with the input finite range D D M 3 Y 1 -Reid potential and the available experimental data at different incident energies. It is shown that a reasonable description of the elastic 12C-12C and 16O-16O scattering data at the low and medium energies can be obtained by using the above LOCV AEI, without any need to define a parametrized density-dependent function in the effective nucleon-nucleon potential, which is formally considered in the typical D D M 3 Y 1 -Reid interactions.

Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1

PubMed Central

Chu, Wen-Ting; Clarke, Jane; Shammas, Sarah L.; Wang, Jin

2017-01-01

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye—Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ionic strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies. PMID:28369057

Alternative modes of client binding enable functional plasticity of Hsp70

NASA Astrophysics Data System (ADS)

Mashaghi, Alireza; Bezrukavnikov, Sergey; Minde, David P.; Wentink, Anne S.; Kityk, Roman; Zachmann-Brand, Beate; Mayer, Matthias P.; Kramer, Günter; Bukau, Bernd; Tans, Sander J.

2016-11-01

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Computational Models of Laryngeal Aerodynamics: Potentials and Numerical Costs.

PubMed

Sadeghi, Hossein; Kniesburges, Stefan; Kaltenbacher, Manfred; Schützenberger, Anne; Döllinger, Michael

2018-02-07

Human phonation is based on the interaction between tracheal airflow and laryngeal dynamics. This fluid-structure interaction is based on the energy exchange between airflow and vocal folds. Major challenges in analyzing the phonatory process in-vivo are the small dimensions and the poor accessibility of the region of interest. For improved analysis of the phonatory process, numerical simulations of the airflow and the vocal fold dynamics have been suggested. Even though most of the models reproduced the phonatory process fairly well, development of comprehensive larynx models is still a subject of research. In the context of clinical application, physiological accuracy and computational model efficiency are of great interest. In this study, a simple numerical larynx model is introduced that incorporates the laryngeal fluid flow. It is based on a synthetic experimental model with silicone vocal folds. The degree of realism was successively increased in separate computational models and each model was simulated for 10 oscillation cycles. Results show that relevant features of the laryngeal flow field, such as glottal jet deflection, develop even when applying rather simple static models with oscillating flow rates. Including further phonatory components such as vocal fold motion, mucosal wave propagation, and ventricular folds, the simulations show phonatory key features like intraglottal flow separation and increased flow rate in presence of ventricular folds. The simulation time on 100 CPU cores ranged between 25 and 290 hours, currently restricting clinical application of these models. Nevertheless, results show high potential of numerical simulations for better understanding of phonatory process. Copyright © 2018 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Systematic analysis of inelastic α scattering off self-conjugate A =4 n nuclei

NASA Astrophysics Data System (ADS)

Adachi, S.; Kawabata, T.; Minomo, K.; Kadoya, T.; Yokota, N.; Akimune, H.; Baba, T.; Fujimura, H.; Fujiwara, M.; Funaki, Y.; Furuno, T.; Hashimoto, T.; Hatanaka, K.; Inaba, K.; Ishii, Y.; Itoh, M.; Iwamoto, C.; Kawase, K.; Maeda, Y.; Matsubara, H.; Matsuda, Y.; Matsuno, H.; Morimoto, T.; Morita, H.; Murata, M.; Nanamura, T.; Ou, I.; Sakaguchi, S.; Sasamoto, Y.; Sawada, R.; Shimizu, Y.; Suda, K.; Tamii, A.; Tameshige, Y.; Tsumura, M.; Uchida, M.; Uesaka, T.; Yoshida, H. P.; Yoshida, S.

2018-01-01

We systematically measured the differential cross sections of inelastic α scattering off self-conjugate A =4 n nuclei at two incident energies Eα=130 MeV and 386 MeV at Research Center for Nuclear Physics, Osaka University. The measured cross sections were analyzed by the distorted-wave Born-approximation (DWBA) calculation using the single-folding potentials, which are obtained by folding macroscopic transition densities with the phenomenological α N interaction. The DWBA calculation with the density-dependent α N interaction systematically overestimates the cross sections for the Δ L =0 transitions. However, the DWBA calculation using the density-independent α N interaction reasonably well describes all the transitions with Δ L =0 -4. We examined uncertainties in the present DWBA calculation stemming from the macroscopic transition densities, distorting potentials, phenomenological α N interaction, and coupled channel effects in 12C. It was found that the DWBA calculation is not sensitive to details of the transition densities nor the distorting potentials, and the phenomenological density-independent α N interaction gives reasonable results. The coupled-channel effects are negligibly small for the 21+ and 31- states in 12C, but not for the 02+ state. However, the DWBA calculation using the density-independent interaction at Eα=386 MeV is still reasonable even for the 02+ state. We concluded that the macroscopic DWBA calculations using the density-independent interaction are reliably applicable to the analysis of inelastic α scattering at Eα˜100 MeV /u .

Energetic frustrations in protein folding at residue resolution: a homologous simulation study of Im9 proteins.

PubMed

Sun, Yunxiang; Ming, Dengming

2014-01-01

Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding.

Study of the Spin Dependent 3HE-NUCLEUS Interaction at 450 Mev

NASA Astrophysics Data System (ADS)

Kamiya, J.; Hatanaka, K.; Sakemi, Y.; Wakasa, T.; Yoshida, H. P.; Obayashi, E.; Hara, K.; Kitamura, K.; Shimizu, Y.; Fujita, K.; Sakamoto, N.; Shimbara, Y.; Adachi, T.; Sakaguchi, H.; Yosoi, M.; Uchida, M.; Yasuda, Y.; Kawabata, T.; Noro, T.

2003-04-01

Differential cross sections and induced polarizations of 3He+12C, 58Ni, and 90Zr elastic scattering were measured at E3He = 450 MeV. This is the first measurement of the polarization for 3He scattering at intermediate energies. The optical potential parameters including the spin-orbit potential were determined with small uncertainties. The volume integrals per nucleon of the potentials were investigated for 3He and their energy dependence showed the similar behavior to that for protons at intermediate energies. The single folding calculations were compared with the data. The real central and spin-orbit parts of the folded potentials had to be reduced by a few tens of percent in order to reproduce the experimental results.

Further optimization of a hybrid united-atom and coarse-grained force field for folding simulations: Improved backbone hydration and interactions between charged side chains

PubMed Central

Han, Wei; Schulten, Klaus

2012-01-01

PACE, a hybrid force field which couples united-atom protein models with coarse-grained (CG) solvent, has been further optimized, aiming to improve itse ciency for folding simulations. Backbone hydration parameters have been re-optimized based on hydration free energies of polyalanyl peptides through atomistic simulations. Also, atomistic partial charges from all-atom force fields were combined with PACE in order to provide a more realistic description of interactions between charged groups. Using replica exchange molecular dynamics (REMD), ab initio folding using the new PACE has been achieved for seven small proteins (16 – 23 residues) with different structural motifs. Experimental data about folded states, such as their stability at room temperature, melting point and NMR NOE constraints, were also well reproduced. Moreover, a systematic comparison of folding kinetics at room temperature has been made with experiments, through standard MD simulations, showing that the new PACE may speed up the actual folding kinetics 5-10 times. Together with the computational speedup benefited from coarse-graining, the force field provides opportunities to study folding mechanisms. In particular, we used the new PACE to fold a 73-residue protein, 3D, in multiple 10 – 30 μs simulations, to its native states (Cα RMSD ~ 0.34 nm). Our results suggest the potential applicability of the new PACE for the study of folding and dynamics of proteins. PMID:23204949

Human Cancer and Platelet Interaction, a Potential Therapeutic Target.

PubMed

Wang, Shike; Li, Zhenyu; Xu, Ren

2018-04-20

Cancer patients experience a four-fold increase in thrombosis risk, indicating that cancer development and progression are associated with platelet activation. Xenograft experiments and transgenic mouse models further demonstrate that platelet activation and platelet-cancer cell interaction are crucial for cancer metastasis. Direct or indirect interaction of platelets induces cancer cell plasticity and enhances survival and extravasation of circulating cancer cells during dissemination. In vivo and in vitro experiments also demonstrate that cancer cells induce platelet aggregation, suggesting that platelet-cancer interaction is bidirectional. Therefore, understanding how platelets crosstalk with cancer cells may identify potential strategies to inhibit cancer metastasis and to reduce cancer-related thrombosis. Here, we discuss the potential function of platelets in regulating cancer progression and summarize the factors and signaling pathways that mediate the cancer cell-platelet interaction.

Identifying Affordances of 3D Printed Tangible Models for Understanding Core Biological Concepts

ERIC Educational Resources Information Center

Davenport, Jodi L.; Silberglitt, Matt; Boxerman, Jonathan; Olson, Arthur

2014-01-01

3D models derived from actual molecular structures have the potential to transform student learning in biology. We share findings related to our research questions: 1) what types of interactions with a protein folding kit promote specific learning objectives?, and 2) what features of the instructional environment (e.g., peer interactions, teacher…

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

PubMed

Bandyopadhyay, Boudhayan; Goldenzweig, Adi; Unger, Tamar; Adato, Orit; Fleishman, Sarel J; Unger, Ron; Horovitz, Amnon

2017-12-15

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A strategy for detecting the conservation of folding-nucleus residues in protein superfamilies.

PubMed

Michnick, S W; Shakhnovich, E

1998-01-01

Nucleation-growth theory predicts that fast-folding peptide sequences fold to their native structure via structures in a transition-state ensemble that share a small number of native contacts (the folding nucleus). Experimental and theoretical studies of proteins suggest that residues participating in folding nuclei are conserved among homologs. We attempted to determine if this is true in proteins with highly diverged sequences but identical folds (superfamilies). We describe a strategy based on comparisons of residue conservation in natural superfamily sequences with simulated sequences (generated with a Monte-Carlo sequence design strategy) for the same proteins. The basic assumptions of the strategy were that natural sequences will conserve residues needed for folding and stability plus function, the simulated sequences contain no functional conservation, and nucleus residues make native contacts with each other. Based on these assumptions, we identified seven potential nucleus residues in ubiquitin superfamily members. Non-nucleus conserved residues were also identified; these are proposed to be involved in stabilizing native interactions. We found that all superfamily members conserved the same potential nucleus residue positions, except those for which the structural topology is significantly different. Our results suggest that the conservation of the nucleus of a specific fold can be predicted by comparing designed simulated sequences with natural highly diverged sequences that fold to the same structure. We suggest that such a strategy could be used to help plan protein folding and design experiments, to identify new superfamily members, and to subdivide superfamilies further into classes having a similar folding mechanism.

All-Atom Four-Body Knowledge-Based Statistical Potentials to Distinguish Native Protein Structures from Nonnative Folds

PubMed Central

2017-01-01

Recent advances in understanding protein folding have benefitted from coarse-grained representations of protein structures. Empirical energy functions derived from these techniques occasionally succeed in distinguishing native structures from their corresponding ensembles of nonnative folds or decoys which display varying degrees of structural dissimilarity to the native proteins. Here we utilized atomic coordinates of single protein chains, comprising a large diverse training set, to develop and evaluate twelve all-atom four-body statistical potentials obtained by exploring alternative values for a pair of inherent parameters. Delaunay tessellation was performed on the atomic coordinates of each protein to objectively identify all quadruplets of interacting atoms, and atomic potentials were generated via statistical analysis of the data and implementation of the inverted Boltzmann principle. Our potentials were evaluated using benchmarking datasets from Decoys-‘R'-Us, and comparisons were made with twelve other physics- and knowledge-based potentials. Ranking 3rd, our best potential tied CHARMM19 and surpassed AMBER force field potentials. We illustrate how a generalized version of our potential can be used to empirically calculate binding energies for target-ligand complexes, using HIV-1 protease-inhibitor complexes for a practical application. The combined results suggest an accurate and efficient atomic four-body statistical potential for protein structure prediction and assessment. PMID:29119109

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

PubMed Central

Ganguly, Debabani; Zhang, Weihong; Chen, Jianhan

2013-01-01

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation. PMID:24278008

Molecular mechanism for the effects of trehalose on beta-hairpin folding revealed by molecular dynamics simulation.

PubMed

Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

2008-11-01

Recent work has shown that trehalose can facilitate and inhibit protein folding, but little is known about the molecular basis of these effects. Molecular-level insights into how the osmolyte affects protein folding are of significance for the rational design of small molecular additives for enhancing or hindering the folding of proteins. To investigate the molecular mechanisms of the facilitation and inhibition effects of trehalose on protein folding, molecular dynamics (MD) simulation of a beta-hairpin peptide (Trp-Arg-Tyr-Tyr-Glu-Ser-Ser-Leu-Glu-Pro-Glu-Pro-Asp) in different trehalose concentrations (0-0.26 mol/L) is performed using an all-atom model. It is found that at a proper trehalose concentration (0.065 mol/L), the peptide folds faster than that in water, but it cannot fold to the beta-hairpin at higher trehalose concentrations. Free energy landscape analysis indicates the presence of three intermediate states in both pure water and in 0.065 mol/L trehalose, but the potential energy barriers in the folding pathway decrease greatly in 0.065 mol/L trehalose, so the peptide folding is facilitated. Moreover, at this trehalose concentration, there is a favorable balance between the peptide backbone hydrogen bonds (H-bonds) and the peptide-trehalose H-bonds, leading to the stabilization of the folded peptide. At higher trehalose concentrations, however, trehalose molecules cluster in the peptide region and interact with the peptide via many H-bonds that prevent the peptide from folding to its native structure. The energy landscape analysis indicates that the potential energy barriers increase so greatly that the peptide cannot overcome it, getting trapped in a local free energy basin. The work reported herein has elucidated the molecular mechanism of the peptide folding in the presence of trehalose.

Short Communication: Inhibition of DC-SIGN-Mediated HIV-1 Infection by Complementary Actions of Dendritic Cell Receptor Antagonists and Env-Targeting Virus Inactivators.

PubMed

Pustylnikov, Sergey; Dave, Rajnish S; Khan, Zafar K; Porkolab, Vanessa; Rashad, Adel A; Hutchinson, Matthew; Fieschi, Frank; Chaiken, Irwin; Jain, Pooja

2016-01-01

The DC-SIGN receptor on human dendritic cells interacts with HIV gp120 to promote both infection of antigen-presenting cells and transinfection of T cells. We hypothesized that in DC-SIGN-expressing cells, both DC-SIGN ligands such as dextrans and gp120 antagonists such as peptide triazoles would inhibit HIV infection with potential complementary antagonist effects. To test this hypothesis, we evaluated the effects of dextran (D66), isomaltooligosaccharides (D06), and several peptide triazoles (HNG156, K13, and UM15) on HIV infection of B-THP-1/DC-SIGN cells. In surface plasmon resonance competition assays, D66 (IC50 = 35.4 μM) and D06 (IC50 = 3.4 mM) prevented binding of soluble DC-SIGN to immobilized mannosylated bovine serum albumin (BSA). An efficacious dose-dependent inhibition of DC-SIGN-mediated HIV infection in both pretreatment and posttreatment settings was observed, as indicated by inhibitory potentials (EC50) [D66 (8 μM), D06 (48 mM), HNG156 (40 μM), UM15 (100 nM), and K13 (25 nM)]. Importantly, both dextrans and peptide triazoles significantly decreased HIV gag RNA levels [D66 (7-fold), D06 (13-fold), HNG156 (7-fold), K-13 (3-fold), and UM15 (6-fold)]. Interestingly, D06 at the highest effective concentration showed a 14-fold decrease of infection, while its combination with 50 μM HNG156 showed a 26-fold decrease. Hence, these compounds can combine to inactivate the viruses and suppress DC-SIGN-mediated virus-cell interaction that as shown earlier leads to dendritic cell HIV infection and transinfection dependent on the DC-SIGN receptor.

Short Communication: Inhibition of DC-SIGN-Mediated HIV-1 Infection by Complementary Actions of Dendritic Cell Receptor Antagonists and Env-Targeting Virus Inactivators

PubMed Central

Pustylnikov, Sergey; Dave, Rajnish S.; Khan, Zafar K.; Porkolab, Vanessa; Rashad, Adel A.; Hutchinson, Matthew; Fieschi, Frank; Chaiken, Irwin

2016-01-01

Abstract The DC-SIGN receptor on human dendritic cells interacts with HIV gp120 to promote both infection of antigen-presenting cells and transinfection of T cells. We hypothesized that in DC-SIGN-expressing cells, both DC-SIGN ligands such as dextrans and gp120 antagonists such as peptide triazoles would inhibit HIV infection with potential complementary antagonist effects. To test this hypothesis, we evaluated the effects of dextran (D66), isomaltooligosaccharides (D06), and several peptide triazoles (HNG156, K13, and UM15) on HIV infection of B-THP-1/DC-SIGN cells. In surface plasmon resonance competition assays, D66 (IC50 = 35.4 μM) and D06 (IC50 = 3.4 mM) prevented binding of soluble DC-SIGN to immobilized mannosylated bovine serum albumin (BSA). An efficacious dose-dependent inhibition of DC-SIGN-mediated HIV infection in both pretreatment and posttreatment settings was observed, as indicated by inhibitory potentials (EC50) [D66 (8 μM), D06 (48 mM), HNG156 (40 μM), UM15 (100 nM), and K13 (25 nM)]. Importantly, both dextrans and peptide triazoles significantly decreased HIV gag RNA levels [D66 (7-fold), D06 (13-fold), HNG156 (7-fold), K-13 (3-fold), and UM15 (6-fold)]. Interestingly, D06 at the highest effective concentration showed a 14-fold decrease of infection, while its combination with 50 μM HNG156 showed a 26-fold decrease. Hence, these compounds can combine to inactivate the viruses and suppress DC-SIGN-mediated virus–cell interaction that as shown earlier leads to dendritic cell HIV infection and transinfection dependent on the DC-SIGN receptor. PMID:26383762

DFMSPH14: A C-code for the double folding interaction potential of two spherical nuclei

NASA Astrophysics Data System (ADS)

Gontchar, I. I.; Chushnyakova, M. V.

2016-09-01

This is a new version of the DFMSPH code designed to obtain the nucleus-nucleus potential by using the double folding model (DFM) and in particular to find the Coulomb barrier. The new version uses the charge, proton, and neutron density distributions provided by the user. Also we added an option for fitting the DFM potential by the Gross-Kalinowski profile. The main functionalities of the original code (e.g. the nucleus-nucleus potential as a function of the distance between the centers of mass of colliding nuclei, the Coulomb barrier characteristics, etc.) have not been modified. Catalog identifier: AEFH_v2_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEFH_v2_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland. Licensing provisions: GNU General Public License, version 3 No. of lines in distributed program, including test data, etc.: 7211 No. of bytes in distributed program, including test data, etc.: 114404 Distribution format: tar.gz Programming language: C Computer: PC and Mac Operation system: Windows XP and higher, MacOS, Unix/Linux Memory required to execute with typical data: below 10 Mbyte Classification: 17.9 Catalog identifier of previous version: AEFH_v1_0 Journal reference of previous version: Comp. Phys. Comm. 181 (2010) 168 Does the new version supersede the previous version?: Yes Nature of physical problem: The code calculates in a semimicroscopic way the bare interaction potential between two colliding spherical nuclei as a function of the center of mass distance. The height and the position of the Coulomb barrier are found. The calculated potential is approximated by an analytical profile (Woods-Saxon or Gross-Kalinowski) near the barrier. Dependence of the barrier parameters upon the characteristics of the effective NN forces (like, e.g. the range of the exchange part of the nuclear term) can be investigated. Method of solution: The nucleus-nucleus potential is calculated using the double folding model with the Coulomb and the effective M3Y NN interactions. For the direct parts of the Coulomb and the nuclear terms, the Fourier transform method is used. In order to calculate the exchange parts, the density matrix expansion method is applied. Typical running time: less than 1 minute. Reason for new version: Many users asked us how to implement their own density distributions in the DFMSPH. Now this option has been added. Also we found that the calculated Double-Folding Potential (DFP) is approximated more accurately by the Gross-Kalinowski (GK) profile. This option has been also added.

Exploring the Universe of Protein Structures beyond the Protein Data Bank

PubMed Central

Cossio, Pilar; Trovato, Antonio; Pietrucci, Fabio; Seno, Flavio; Maritan, Amos; Laio, Alessandro

2010-01-01

It is currently believed that the atlas of existing protein structures is faithfully represented in the Protein Data Bank. However, whether this atlas covers the full universe of all possible protein structures is still a highly debated issue. By using a sophisticated numerical approach, we performed an exhaustive exploration of the conformational space of a 60 amino acid polypeptide chain described with an accurate all-atom interaction potential. We generated a database of around 30,000 compact folds with at least of secondary structure corresponding to local minima of the potential energy. This ensemble plausibly represents the universe of protein folds of similar length; indeed, all the known folds are represented in the set with good accuracy. However, we discover that the known folds form a rather small subset, which cannot be reproduced by choosing random structures in the database. Rather, natural and possible folds differ by the contact order, on average significantly smaller in the former. This suggests the presence of an evolutionary bias, possibly related to kinetic accessibility, towards structures with shorter loops between contacting residues. Beside their conceptual relevance, the new structures open a range of practical applications such as the development of accurate structure prediction strategies, the optimization of force fields, and the identification and design of novel folds. PMID:21079678

In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL.

PubMed

Kumar, Vipul; Punetha, Ankita; Sundar, Durai; Chaudhuri, Tapan K

2012-01-01

Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. They bind newly synthesized or non-native polypeptides through hydrophobic interactions and prevent their aggregation. Some proteins do not interact with GroEL, hence even though they are aggregation prone, cannot be assisted by GroEL for their folding. In this study, we have attempted to engineer these non-substrate proteins to convert them as the substrate for GroEL, without compromising on their function. We have used a computational biology approach to generate mutants of the selected proteins by selectively mutating residues in the hydrophobic patch, similar to GroES mobile loop region that are responsible for interaction with GroEL, and compared with the wild counterparts for calculation of their instability and aggregation propensities. The energies of the newly designed mutants were computed through molecular dynamics simulations. We observed increased aggregation propensity of some of the mutants formed after replacing charged amino acid residues with hydrophobic ones in the well defined hydrophobic patch, raising the possibility of their binding ability to GroEL. The newly generated mutants may provide potential substrates for Chaperonin GroEL, which can be experimentally generated and tested for their tendency of aggregation, interactions with GroEL and the possibility of chaperone-assisted folding to produce functional proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near positionmore » 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.« less

The PYRIN domain: A member of the death domain-fold superfamily

PubMed Central

Fairbrother, Wayne J.; Gordon, Nathaniel C.; Humke, Eric W.; O'Rourke, Karen M.; Starovasnik, Melissa A.; Yin, Jian-Ping; Dixit, Vishva M.

2001-01-01

PYRIN domains were identified recently as putative protein–protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The ∼95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein–protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction. PMID:11514682

FE Modelling of the Fluid-Structure-Acoustic Interaction for the Vocal Folds Self-Oscillation

NASA Astrophysics Data System (ADS)

Švancara, Pavel; Horáček, J.; Hrůza, V.

The flow induced self-oscillation of the human vocal folds in interaction with acoustic processes in the simplified vocal tract model was explored by three-dimensional (3D) finite element (FE) model. Developed FE model includes vocal folds pretension before phonation, large deformations of the vocal fold tissue, vocal folds contact, fluid-structure interaction, morphing the fluid mesh according the vocal folds motion (Arbitrary Lagrangian-Eulerian approach), unsteady viscous compressible airflow described by the Navier-Stokes equations and airflow separation during the glottis closure. Iterative partitioned approach is used for modelling the fluid-structure interaction. Computed results prove that the developed model can be used for simulation of the vocal folds self-oscillation and resulting acoustic waves. The developed model enables to numerically simulate an influence of some pathological changes in the vocal fold tissue on the voice production.

Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

PubMed

Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

2013-12-23

Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²⁺ bimetallo core.

Regulatory effects of cotranscriptional RNA structure formation and transitions.

PubMed

Liu, Sheng-Rui; Hu, Chun-Gen; Zhang, Jin-Zhi

2016-09-01

RNAs, which play significant roles in many fundamental biological processes of life, fold into sophisticated and precise structures. RNA folding is a dynamic and intricate process, which conformation transition of coding and noncoding RNAs form the primary elements of genetic regulation. The cellular environment contains various intrinsic and extrinsic factors that potentially affect RNA folding in vivo, and experimental and theoretical evidence increasingly indicates that the highly flexible features of the RNA structure are affected by these factors, which include the flanking sequence context, physiochemical conditions, cis RNA-RNA interactions, and RNA interactions with other molecules. Furthermore, distinct RNA structures have been identified that govern almost all steps of biological processes in cells, including transcriptional activation and termination, transcriptional mutagenesis, 5'-capping, splicing, 3'-polyadenylation, mRNA export and localization, and translation. Here, we briefly summarize the dynamic and complex features of RNA folding along with a wide variety of intrinsic and extrinsic factors that affect RNA folding. We then provide several examples to elaborate RNA structure-mediated regulation at the transcriptional and posttranscriptional levels. Finally, we illustrate the regulatory roles of RNA structure and discuss advances pertaining to RNA structure in plants. WIREs RNA 2016, 7:562-574. doi: 10.1002/wrna.1350 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

High-lying intermediate excitations in the nuclear effective interaction with a super-soft-core potential

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goode, P.R.; Barrett, B.R.; Portilho, O.

1979-02-01

The earlier calculations of Goode and Barrett are repeated using the super-soft-core potential of Gogny, Pires, and de Tourreil. The particular third-order folded diagram which they calculated now converges in its intermediate-state energy summation, because of the suppression of the strong short-range repulsive effects present in earlier calculations.

Semimicroscopic, Lane-consistent nucleon-nucleus optical model potential up to 200 MeV

NASA Astrophysics Data System (ADS)

Bauge, Eric; Delaroche, Jean-Paul; Girod, Michel

2000-10-01

Our semimicroscopic optical model potential (E. Bauge et al., Phys. Rev. C 58), 1118 (1998). is re-evaluated in order to obtain a Lane-consistent description of (p,p), (n,n) and (p,n IAS) elastic scattering and reaction observables. The re-assessed nuclear matter interaction (which includes sizable renormalizations of the isovector potentials) is folded with microscopic HFB nuclear densities, producing OMPs that are free of adjustable parameters for nuclei with A >= 40. With Lane-consistency of the interaction, and the predictive nature of our HFB calculations, this scheme can be used to calculate observables for nuclei far from the stability line with good predictivity.

In vitro interactions with repeated grapefruit juice administration--to peel or not to peel?

PubMed

Brill, Shlomo; Zimmermann, Christian; Berger, Karin; Drewe, Juergen; Gutmann, Heike

2009-03-01

Interactions of acutely administered grapefruit juice (GFJ) with cytochrome P450 isoform 3A4 (CYP3A4) and P-glycoprotein (Pgp) function are well established. In this study, we investigated in vitro the effect of repeated administration of GFJ and its major constituents (the flavonoid naringin, its aglycone naringenin and the furanocoumarin bergamottin) on mRNA expression of MDR1 and CYP3A4 in LS180 cells. Since the bergamottin content is higher in the peel than in the fruit, we compared GFJ containing peel (GFJP+) with juice without any peel extract (GFJP-). GFJP- (1%) showed no significant effect on MDR1 and CYP3A4 mRNA expression, whereas 1% GFJP+ increased expression of MDR1 3.7-fold (P<0.01) and CYP3A4 2.3-fold (P<0.05). Of the tested constituents, only 10 microM bergamottin and 200 microM naringenin induced MDR1 mRNA levels 2.9- and 4.0-fold, respectively (P<0.01 for both), and CYP3A4 mRNA levels 3.2- and 15.6-fold (P<0.01 for both), respectively. Western blot analysis and rhodamine 123 uptake experiments partly confirmed these findings on the protein and the functional level. In summary, GFJ containing no peel extract may have a lower potential for interactions with CYP3A4 or P-glycoprotein.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

NASA Astrophysics Data System (ADS)

Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

2018-05-01

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain.

PubMed

Pavani, R S; Fernandes, C; Perez, A M; Vasconcelos, E J R; Siqueira-Neto, J L; Fontes, M R; Cano, M I N

2014-12-20

Replication protein A-1 (RPA-1) is a single-stranded DNA-binding protein involved in DNA metabolism. We previously demonstrated the interaction between LaRPA-1 and telomeric DNA. Here, we expressed and purified truncated mutants of LaRPA-1 and used circular dichroism measurements and molecular dynamics simulations to demonstrate that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1. LaRPA-1 interacts with telomeric ssDNA via its N-terminal OB-fold domain, whereas RPA from higher eukaryotes show different binding modes to ssDNA. Our results show that LaRPA-1 is evolutionary distinct from other RPA-1 proteins and can potentially be used for targeting trypanosomatid telomeres. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Polymer physics predicts the effects of structural variants on chromatin architecture.

PubMed

Bianco, Simona; Lupiáñez, Darío G; Chiariello, Andrea M; Annunziatella, Carlo; Kraft, Katerina; Schöpflin, Robert; Wittler, Lars; Andrey, Guillaume; Vingron, Martin; Pombo, Ana; Mundlos, Stefan; Nicodemi, Mario

2018-05-01

Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.

RNA 3D Structural Motifs: Definition, Identification, Annotation, and Database Searching

NASA Astrophysics Data System (ADS)

Nasalean, Lorena; Stombaugh, Jesse; Zirbel, Craig L.; Leontis, Neocles B.

Structured RNA molecules resemble proteins in the hierarchical organization of their global structures, folding and broad range of functions. Structured RNAs are composed of recurrent modular motifs that play specific functional roles. Some motifs direct the folding of the RNA or stabilize the folded structure through tertiary interactions. Others bind ligands or proteins or catalyze chemical reactions. Therefore, it is desirable, starting from the RNA sequence, to be able to predict the locations of recurrent motifs in RNA molecules. Conversely, the potential occurrence of one or more known 3D RNA motifs may indicate that a genomic sequence codes for a structured RNA molecule. To identify known RNA structural motifs in new RNA sequences, precise structure-based definitions are needed that specify the core nucleotides of each motif and their conserved interactions. By comparing instances of each recurrent motif and applying base pair isosteriCity relations, one can identify neutral mutations that preserve its structure and function in the contexts in which it occurs.

Simulating protein folding initiation sites using an alpha-carbon-only knowledge-based force field

PubMed Central

Buck, Patrick M.; Bystroff, Christopher

2015-01-01

Protein folding is a hierarchical process where structure forms locally first, then globally. Some short sequence segments initiate folding through strong structural preferences that are independent of their three-dimensional context in proteins. We have constructed a knowledge-based force field in which the energy functions are conditional on local sequence patterns, as expressed in the hidden Markov model for local structure (HMMSTR). Carbon-alpha force field (CALF) builds sequence specific statistical potentials based on database frequencies for α-carbon virtual bond opening and dihedral angles, pairwise contacts and hydrogen bond donor-acceptor pairs, and simulates folding via Brownian dynamics. We introduce hydrogen bond donor and acceptor potentials as α-carbon probability fields that are conditional on the predicted local sequence. Constant temperature simulations were carried out using 27 peptides selected as putative folding initiation sites, each 12 residues in length, representing several different local structure motifs. Each 0.6 μs trajectory was clustered based on structure. Simulation convergence or representativeness was assessed by subdividing trajectories and comparing clusters. For 21 of the 27 sequences, the largest cluster made up more than half of the total trajectory. Of these 21 sequences, 14 had cluster centers that were at most 2.6 Å root mean square deviation (RMSD) from their native structure in the corresponding full-length protein. To assess the adequacy of the energy function on nonlocal interactions, 11 full length native structures were relaxed using Brownian dynamics simulations. Equilibrated structures deviated from their native states but retained their overall topology and compactness. A simple potential that folds proteins locally and stabilizes proteins globally may enable a more realistic understanding of hierarchical folding pathways. PMID:19137613

Cooperative Tertiary Interaction Network Guides RNA Folding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Behrouzi, Reza; Roh, Joon Ho; Kilburn, Duncan

2013-04-08

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends onmore » the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.« less

Validity of Gō models: comparison with a solvent-shielded empirical energy decomposition.

PubMed

Paci, Emanuele; Vendruscolo, Michele; Karplus, Martin

2002-12-01

Do Gō-type model potentials provide a valid approach for studying protein folding? They have been widely used for this purpose because of their simplicity and the speed of simulations based on their use. The essential assumption in such models is that only contact interactions existing in the native state determine the energy surface of a polypeptide chain, even for non-native configurations sampled along folding trajectories. Here we use an all-atom molecular mechanics energy function to investigate the adequacy of Gō-type potentials. We show that, although the contact approximation is accurate, non-native contributions to the energy can be significant. The assumed relation between residue-residue interaction energies and the number of contacts between them is found to be only approximate. By contrast, individual residue energies correlate very well with the number of contacts. The results demonstrate that models based on the latter should give meaningful results (e.g., as used to interpret phi values), whereas those that depend on the former are only qualitative, at best.

Anisotropy of the Coulomb Interaction between Folded Proteins: Consequences for Mesoscopic Aggregation of Lysozyme

PubMed Central

Chan, Ho Yin; Lankevich, Vladimir; Vekilov, Peter G.; Lubchenko, Vassiliy

2012-01-01

Toward quantitative description of protein aggregation, we develop a computationally efficient method to evaluate the potential of mean force between two folded protein molecules that allows for complete sampling of their mutual orientation. Our model is valid at moderate ionic strengths and accounts for the actual charge distribution on the surface of the molecules, the dielectric discontinuity at the protein-solvent interface, and the possibility of protonation or deprotonation of surface residues induced by the electric field due to the other protein molecule. We apply the model to the protein lysozyme, whose solutions exhibit both mesoscopic clusters of protein-rich liquid and liquid-liquid separation; the former requires that protein form complexes with typical lifetimes of approximately milliseconds. We find the electrostatic repulsion is typically lower than the prediction of the Derjaguin-Landau-Verwey-Overbeek theory. The Coulomb interaction in the lowest-energy docking configuration is nonrepulsive, despite the high positive charge on the molecules. Typical docking configurations barely involve protonation or deprotonation of surface residues. The obtained potential of mean force between folded lysozyme molecules is consistent with the location of the liquid-liquid coexistence, but produces dimers that are too short-lived for clusters to exist, suggesting lysozyme undergoes conformational changes during cluster formation. PMID:22768950

Investigating the importance of Delaunay-based definition of atomic interactions in scoring of protein-protein docking results.

PubMed

Jafari, Rahim; Sadeghi, Mehdi; Mirzaie, Mehdi

2016-05-01

The approaches taken to represent and describe structural features of the macromolecules are of major importance when developing computational methods for studying and predicting their structures and interactions. This study attempts to explore the significance of Delaunay tessellation for the definition of atomic interactions by evaluating its impact on the performance of scoring protein-protein docking prediction. Two sets of knowledge-based scoring potentials are extracted from a training dataset of native protein-protein complexes. The potential of the first set is derived using atomic interactions extracted from Delaunay tessellated structures. The potential of the second set is calculated conventionally, that is, using atom pairs whose interactions were determined by their separation distances. The scoring potentials were tested against two different docking decoy sets and their performances were compared. The results show that, if properly optimized, the Delaunay-based scoring potentials can achieve higher success rate than the usual scoring potentials. These results and the results of a previous study on the use of Delaunay-based potentials in protein fold recognition, all point to the fact that Delaunay tessellation of protein structure can provide a more realistic definition of atomic interaction, and therefore, if appropriately utilized, may be able to improve the accuracy of pair potentials. Copyright © 2016 Elsevier Inc. All rights reserved.

Insights into the fold organization of TIM barrel from interaction energy based structure networks.

PubMed

Vijayabaskar, M S; Vishveshwara, Saraswathi

2012-01-01

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

Dose-dependent induction of cytochrome P450 (CYP) 3A4 and activation of pregnane X receptor by topiramate.

PubMed

Nallani, Srikanth C; Glauser, Tracy A; Hariparsad, Niresh; Setchell, Kenneth; Buckley, Donna J; Buckley, Arthur R; Desai, Pankaj B

2003-12-01

In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation. The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.

Conformational transition free energy profiles of an adsorbed, lattice model protein by multicanonical Monte Carlo simulation

NASA Astrophysics Data System (ADS)

Castells, Victoria; Van Tassel, Paul R.

2005-02-01

Proteins often undergo changes in internal conformation upon interacting with a surface. We investigate the thermodynamics of surface induced conformational change in a lattice model protein using a multicanonical Monte Carlo method. The protein is a linear heteropolymer of 27 segments (of types A and B) confined to a cubic lattice. The segmental order and nearest neighbor contact energies are chosen to yield, in the absence of an adsorbing surface, a unique 3×3×3 folded structure. The surface is a plane of sites interacting either equally with A and B segments (equal affinity surface) or more strongly with the A segments (A affinity surface). We use a multicanonical Monte Carlo algorithm, with configuration bias and jump walking moves, featuring an iteratively updated sampling function that converges to the reciprocal of the density of states 1/Ω(E), E being the potential energy. We find inflection points in the configurational entropy, S(E)=klnΩ(E), for all but a strongly adsorbing equal affinity surface, indicating the presence of free energy barriers to transition. When protein-surface interactions are weak, the free energy profiles F(E)=E-TS(E) qualitatively resemble those of a protein in the absence of a surface: a free energy barrier separates a folded, lowest energy state from globular, higher energy states. The surface acts in this case to stabilize the globular states relative to the folded state. When the protein surface interactions are stronger, the situation differs markedly: the folded state no longer occurs at the lowest energy and free energy barriers may be absent altogether.

Unique role of ionic liquid [bmin][BF 4] during curcumin-surfactant association and micellization of cationic, anionic and non-ionic surfactant solutions

NASA Astrophysics Data System (ADS)

Patra, Digambara; Barakat, Christelle

2011-09-01

Hydrophilic ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroburate, modified the properties of aqueous surfactant solutions associated with curcumin. Because of potential pharmaceutical applications as an antioxidant, anti-inflammatory and anti-carcinogenic agent, curcumin has received ample attention as potential drug. The interaction of curcumin with various charged aqueous surfactant solutions showed it exists in deprotonated enol form in surfactant solutions. The nitro and hydroxyl groups of o-nitrophenol interact with the carbonyl and hydroxyl groups of the enol form of curcumin by forming ground state complex through hydrogen bonds and offered interesting information about the nature of the interactions between the aqueous surfactant solutions and curcumin depending on charge of head group of the surfactant. IL[bmin][BF 4] encouraged early formation of micelle in case of cationic and anionic aqueous surfactant solutions, but slightly prolonged micelle formation in the case of neutral aqueous surfactant solution. However, for curcumin IL [bmin][BF 4] favored strong association (7-fold increase) with neutral surfactant solution, marginally supported association with anionic surfactant solution and discouraged (˜2-fold decrease) association with cationic surfactant solution.

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

PubMed Central

An, Na; Fleming, Aaron M.; Middleton, Eric G.; Burrows, Cynthia J.

2014-01-01

Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent electrical signatures through DNA–protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology. PMID:25225404

Modeling coupled aerodynamics and vocal fold dynamics using immersed boundary methods.

PubMed

Duncan, Comer; Zhai, Guangnian; Scherer, Ronald

2006-11-01

The penalty immersed boundary (PIB) method, originally introduced by Peskin (1972) to model the function of the mammalian heart, is tested as a fluid-structure interaction model of the closely coupled dynamics of the vocal folds and aerodynamics in phonation. Two-dimensional vocal folds are simulated with material properties chosen to result in self-oscillation and volume flows in physiological frequency ranges. Properties of the glottal flow field, including vorticity, are studied in conjunction with the dynamic vocal fold motion. The results of using the PIB method to model self-oscillating vocal folds for the case of 8 cm H20 as the transglottal pressure gradient are described. The volume flow at 8 cm H20, the transglottal pressure, and vortex dynamics associated with the self-oscillating model are shown. Volume flow is also given for 2, 4, and 12 cm H2O, illustrating the robustness of the model to a range of transglottal pressures. The results indicate that the PIB method applied to modeling phonation has good potential for the study of the interdependence of aerodynamics and vocal fold motion.

The influence of metoprolol dosage release formulation on the pharmacokinetic drug interaction with paroxetine

PubMed Central

Stout, Stephen M.; Nielsen, Jace; Welage, Lynda S.; Shea, Michael; Brook, Robert; Kerber, Kevin; Bleske, Barry E.

2010-01-01

Studies have demonstrated an influence of dosage release formulations on drug interactions and enantiomeric plasma concentrations. Metoprolol is a commonly used β-adrenergic antagonist metabolized by CYP2D6. The CYP2D6 inhibitor paroxetine has previously been shown to interact with metoprolol tartrate. This open-label, randomized, 4 phase crossover study assessed the potential differential effects of paroxetine on stereoselective pharmacokinetics of immediate release (IR) tartrate and extended release (ER) succinate metoprolol formulations. Ten healthy subjects received metoprolol IR (50 mg) and ER (100 mg) with and without paroxetine coadministration. Blood samples were collected over 24 hours for determination of metoprolol plasma enantiomer concentrations. Paroxetine coadministration significantly increased S and R metoprolol AUC0–24h by 4 and 5 fold, respectively for IR, and 3 and 4 fold, respectively for ER. S/R AUC ratios significantly decreased. These results demonstrate a pharmacokinetic interaction between paroxetine and both formulations of metoprolol. The interaction is greater with R metoprolol and stereoselective metabolism is lost. This could theoretically result in greater β-blockade and lost cardioselectivity. The magnitude of the interaction was similar between metoprolol formulations, which may be attributable to low doses / drug input rates employed. PMID:20400652

Evaluation of Drug-Drug Interaction Potential Between Sacubitril/Valsartan (LCZ696) and Statins Using a Physiologically Based Pharmacokinetic Model.

PubMed

Lin, Wen; Ji, Tao; Einolf, Heidi; Ayalasomayajula, Surya; Lin, Tsu-Han; Hanna, Imad; Heimbach, Tycho; Breen, Christopher; Jarugula, Venkateswar; He, Handan

2017-05-01

Sacubitril/valsartan (LCZ696) has been approved for the treatment of heart failure. Sacubitril is an in vitro inhibitor of organic anion-transporting polypeptides (OATPs). In clinical studies, LCZ696 increased atorvastatin C max by 1.7-fold and area under the plasma concentration-time curve by 1.3-fold, but had little or no effect on simvastatin or simvastatin acid exposure. A physiologically based pharmacokinetics modeling approach was applied to explore the underlying mechanisms behind the statin-specific LCZ696 drug interaction observations. The model incorporated OATP-mediated clearance (CL int,T ) for simvastatin and simvastatin acid to successfully describe the pharmacokinetic profiles of either analyte in the absence or presence of LCZ696. Moreover, the model successfully described the clinically observed drug effect with atorvastatin. The simulations clarified the critical parameters responsible for the observation of a low, yet clinically relevant, drug-drug interaction DDI between sacubitril and atorvastatin and the lack of effect with simvastatin acid. Atorvastatin is administered in its active form and rapidly achieves C max that coincide with the low C max of sacubitril. In contrast, simvastatin requires a hydrolysis step to the acid form and therefore is not present at the site of interactions at sacubitril concentrations that are inhibitory. Similar models were used to evaluate the drug-drug interaction risk for additional OATP-transported statins which predicted to maximally result in a 1.5-fold exposure increase. Copyright © 2017. Published by Elsevier Inc.

Transmembrane Segments Form Tertiary Hairpins in the Folding Vestibule of the Ribosome.

PubMed Central

Tu, LiWei; Khanna, Pooja; Deutsch, Carol

2013-01-01

Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100 Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a ‘folding vestibule’ that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains, but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved, intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin (‘paddle’) of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane. PMID:24055377

Designing pH induced fold switch in proteins

NASA Astrophysics Data System (ADS)

Baruah, Anupaul; Biswas, Parbati

2015-05-01

This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

PubMed

Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

2015-05-22

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

PubMed Central

Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

2015-01-01

Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance. Conclusions/Significance We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity. Trial Registration ClinicalTrials.gov NCT00663546 PMID:26701602

Inversion of the Balance between Hydrophobic and Hydrogen Bonding Interactions in Protein Folding and Aggregation

PubMed Central

Fitzpatrick, Anthony W.; Knowles, Tuomas P. J.; Waudby, Christopher A.; Vendruscolo, Michele; Dobson, Christopher M.

2011-01-01

Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions. PMID:22022239

On topological RNA interaction structures.

PubMed

Qin, Jing; Reidys, Christian M

2013-07-01

Recently a folding algorithm of topological RNA pseudoknot structures was presented in Reidys et al. (2011). This algorithm folds single-stranded γ-structures, that is, RNA structures composed by distinct motifs of bounded topological genus. In this article, we set the theoretical foundations for the folding of the two backbone analogues of γ structures: the RNA γ-interaction structures. These are RNA-RNA interaction structures that are constructed by a finite number of building blocks over two backbones having genus at most γ. Combinatorial properties of γ-interaction structures are of practical interest since they have direct implications for the folding of topological interaction structures. We compute the generating function of γ-interaction structures and show that it is algebraic, which implies that the numbers of interaction structures can be computed recursively. We obtain simple asymptotic formulas for 0- and 1-interaction structures. The simplest class of interaction structures are the 0-interaction structures, which represent the two backbone analogues of secondary structures.

A residue in helical conformation in the native state adopts a β-strand conformation in the folding transition state despite its high and canonical Φ-value.

PubMed

Zarrine-Afsar, Arash; Dahesh, Samira; Davidson, Alan R

2012-05-01

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state. Copyright © 2012 Wiley Periodicals, Inc.

Interactions of microbicide nanoparticles with a simulated vaginal fluid.

PubMed

das Neves, José; Rocha, Cristina M R; Gonçalves, Maria Pilar; Carrier, Rebecca L; Amiji, Mansoor; Bahia, Maria Fernanda; Sarmento, Bruno

2012-11-05

The interaction with cervicovaginal mucus presents the potential to impact the performance of drug nanocarriers. These systems must migrate through this biological fluid in order to deliver their drug payload to the underlying mucosal surface. We studied the ability of dapivirine-loaded polycaprolactone (PCL)-based nanoparticles (NPs) to interact with a simulated vaginal fluid (SVF) incorporating mucin. Different surface modifiers were used to produce NPs with either negative (poloxamer 338 NF and sodium lauryl sulfate) or positive (cetyltrimethylammonium bromide) surface charge. Studies were performed using the mucin particle method, rheological measurements, and real-time multiple particle tracking. Results showed that SVF presented rheological properties similar to those of human cervicovaginal mucus. Analysis of NP transport indicated mild interactions with mucin and low adhesive potential. In general, negatively charged NPs underwent subdiffusive transport in SVF, i.e., hindered as compared to their diffusion in water, but faster than for positively charged NPs. These differences were increased when the pH of SVF was changed from 4.2 to 7.0. Diffusivity was 50- and 172-fold lower in SVF at pH 4.2 than in water for negatively charged and positively charged NPs, respectively. At pH 7.0, this decrease was around 20- and 385-fold, respectively. The estimated times required to cross a layer of SVF were equal to or lower than 1.7 h for negatively charged NPs, while for positively charged NPs these values were equal to or higher than 7 h. Overall, our results suggest that negatively charged PCL NPs may be suitable to be used as carriers in order to deliver dapivirine and potentially other antiretroviral drugs to the cervicovaginal mucosal lining. Also, they further reinforce the importance in characterizing the interactions of nanosystems with mucus fluids or surrogates when considering mucosal drug delivery.

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

PubMed Central

Sengupta, Raghuvir N.; Van Schie, Sabine N.S.; Giambaşu, George; Dai, Qing; Yesselman, Joseph D.; York, Darrin; Piccirilli, Joseph A.; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

PubMed

Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. © 2015 Sengupta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Effect of charged amino acid side chain length on lateral cross-strand interactions between carboxylate- and guanidinium-containing residues in a β-hairpin.

PubMed

Kuo, Hsiou-Ting; Liu, Shing-Lung; Chiu, Wen-Chieh; Fang, Chun-Jen; Chang, Hsien-Chen; Wang, Wei-Ren; Yang, Po-An; Li, Jhe-Hao; Huang, Shing-Jong; Huang, Shou-Ling; Cheng, Richard P

2015-05-01

β-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, β-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the β-hairpin motif.

Evaluating Protein Structure and Dynamics Using Co-Solvents, Photochemical Triggers, and Site-Specific Spectroscopic Probes

NASA Astrophysics Data System (ADS)

Abaskharon, Rachel M.

As ubiquitous and diverse biopolymers, proteins are dynamic molecules that are constantly engaging in inter- and intramolecular interactions responsible for their structure, fold, and function. Because of this, gaining a comprehensive understanding of the factors that control protein conformation and dynamics remains elusive as current experimental techniques often lack the ability to initiate and probe a specific interaction or conformational transition. For this reason, this thesis aims to develop methods to control and monitor protein conformations, conformational transitions, and dynamics in a site-specific manner, as well as to understand how specific and non-specific interactions affect the protein folding energy landscape. First, by using the co-solvent, trifluoroethanol (TFE), we show that the rate at which a peptide folds can be greatly impacted and thus controlled by the excluded volume effect. Secondly, we demonstrate the utility of several light-responsive molecules and reactions as methods to manipulate and investigate protein-folding processes. Using an azobenzene linker as a photo-initiator, we are able to increase the folding rate of a protein system by an order of magnitude by channeling a sub-population through a parallel, faster folding pathway. Additionally, we utilize a tryptophan-mediated electron transfer process to a nearby disulfide bond to strategically unfold a protein molecule with ultraviolet light. We also demonstrate the potential of two ruthenium polypyridyl complexes as ultrafast phototriggers of protein reactions. Finally, we develop several site-specific spectroscopic probes of protein structure and environment. Specifically, we demonstrate that a 13C-labeled aspartic acid residue constitutes a useful site-specific infrared probe for investigating salt-bridges and hydration dynamics of proteins, particularly in proteins containing several acidic amino acids. We also show that a proline-derivative, 4-oxoproline, possesses novel infrared properties that can be exploited to monitor the cis-trans isomerization process of individual proline residues in proteins.

A proposed OB-fold with a protein-interaction surface in Candida albicans telomerase protein Est3

PubMed Central

Yu, Eun Young; Wang, Feng; Lei, Ming; Lue, Neal F

2008-01-01

Ever shorter telomeres 3 (Est3) is an essential telomerase regulatory subunit thought to be unique to budding yeasts. Here we use multiple sequence alignment and hidden Markov model–hidden Markov model (HMM-HMM) comparison to uncover potential similarities between Est3 and the mammalian telomeric protein Tpp1. Analysis of site-specific mutants of Candida albicans Est3 revealed functional distinctions between residues that are conserved between Est3 and Tpp1 and those that are unique to Est3. Although both types of residues are important for telomere maintenance in vivo, only the former contributes to telomerase activity in vitro and facilitates the association of Est3 with telomerase core components. Consistent with a function in protein-protein interaction, the residues common to Est3 and Tpp1 map to one face of an OB-fold model structure, away from the canonical nucleic acid binding surface. We propose that Est3 and the OB-fold domain of Tpp1 mediate a conserved function in telomerase regulation. PMID:19172753

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

PubMed Central

Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

2008-01-01

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

A Systematic Approach to Evaluate Herb-Drug Interaction Mechanisms: Investigation of Milk Thistle Extracts and Eight Isolated Constituents as CYP3A Inhibitors

PubMed Central

Brantley, Scott J.; Graf, Tyler N.; Oberlies, Nicholas H.

2013-01-01

Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 1′-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 μM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC50 determination. The IC50s for isosilybin B and silychristin were ∼60 and 90 μM, respectively, whereas those for the remaining constituents were >100 μM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (KI, ∼100 μM; kinact, ∼0.20 min−1) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation. PMID:23801821

A systematic approach to evaluate herb-drug interaction mechanisms: investigation of milk thistle extracts and eight isolated constituents as CYP3A inhibitors.

PubMed

Brantley, Scott J; Graf, Tyler N; Oberlies, Nicholas H; Paine, Mary F

2013-09-01

Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 1'-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 μM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC50 determination. The IC50s for isosilybin B and silychristin were ∼60 and 90 μM, respectively, whereas those for the remaining constituents were >100 μM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (KI, ∼100 μM; kinact, ∼0.20 min(-1)) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation.

Potentiation of the bioavailability of blueberry phenolic compounds by co-ingested grape phenolic compounds in mice, revealed by targeted metabolomic profiling in plasma and feces.

PubMed

Dudonné, Stéphanie; Dal-Pan, Alexandre; Dubé, Pascal; Varin, Thibault V; Calon, Frédéric; Desjardins, Yves

2016-08-10

The low bioavailability of dietary phenolic compounds, resulting from poor absorption and high rates of metabolism and excretion, is a concern as it can limit their potential beneficial effects on health. Targeted metabolomic profiling in plasma and feces of mice supplemented for 15 days with a blueberry extract, a grape extract or their combination revealed significantly increased plasma concentrations (3-5 fold) of blueberry phenolic metabolites in the presence of a co-ingested grape extract, associated with an equivalent decrease in their appearance in feces. Additionally, the repeated daily administration of the blueberry-grape combination significantly increased plasma phenolic concentrations (2-3-fold) compared to animals receiving only a single acute dose, with no such increase being observed with individual extracts. These findings highlight a positive interaction between blueberry and grape constituents, in which the grape extract enhanced the absorption of blueberry phenolic compounds. This study provides for the first time in vivo evidence of such an interaction occurring between co-ingested phenolic compounds from fruit extracts leading to their improved bioavailability.

Effect of interactions with the chaperonin cavity on protein folding and misfolding†

PubMed Central

Sirur, Anshul; Knott, Michael; Best, Robert B.

2015-01-01

Recent experimental and computational results have suggested that attractive interactions between a chaperonin and an enclosed substrate can have an important effect on the protein folding rate: it appears that folding may even be slower inside the cavity than under unconfined conditions, in contrast to what we would expect from excluded volume effects on the unfolded state. Here we examine systematically the dependence of the protein stability and folding rate on the strength of such attractive interactions between the chaperonin and substrate, by using molecular simulations of model protein systems in an idealised attractive cavity. Interestingly, we find a maximum in stability, and a rate which indeed slows down at high attraction strengths. We have developed a simple phenomenological model which can explain the variations in folding rate and stability due to differing effects on the free energies of the unfolded state, folded state, and transition state; changes in the diffusion coefficient along the folding coordinate are relatively small, at least for our simplified model. In order to investigate a possible role for these attractive interactions in folding, we have studied a recently developed model for misfolding in multidomain proteins. We find that, while encapsulation in repulsive cavities greatly increases the fraction of misfolded protein, sufficiently strong attractive protein-cavity interactions can strongly reduce the fraction of proteins reaching misfolded traps. PMID:24077053

Importance of the ion-pair interactions in the OPEP coarse-grained force field: parametrization and validation.

PubMed

Sterpone, Fabio; Nguyen, Phuong H; Kalimeri, Maria; Derreumaux, Philippe

2013-10-08

We have derived new effective interactions that improve the description of ion-pairs in the OPEP coarse-grained force field without introducing explicit electrostatic terms. The iterative Boltzmann inversion method was used to extract these potentials from all atom simulations by targeting the radial distribution function of the distance between the center of mass of the side-chains. The new potentials have been tested on several systems that differ in structural properties, thermodynamic stabilities and number of ion-pairs. Our modeling, by refining the packing of the charged amino-acids, impacts the stability of secondary structure motifs and the population of intermediate states during temperature folding/unfolding; it also improves the aggregation propensity of peptides. The new version of the OPEP force field has the potentiality to describe more realistically a large spectrum of situations where salt-bridges are key interactions.

Internalization of Red Blood Cell-Mimicking Hydrogel Capsules with pH-Triggered Shape Responses

PubMed Central

2015-01-01

We report on naturally inspired hydrogel capsules with pH-induced transitions from discoids to oblate ellipsoids and their interactions with cells. We integrate characteristics of erythrocytes such as discoidal shape, hollow structure, and elasticity with reversible pH-responsiveness of poly(methacrylic acid) (PMAA) to design a new type of drug delivery carrier to be potentially triggered by chemical stimuli in the tumor lesion. The capsules are fabricated from cross-linked PMAA multilayers using sacrificial discoid silicon templates. The degree of capsule shape transition is controlled by the pH-tuned volume change, which in turn is regulated by the capsule wall composition. The (PMAA)15 capsules undergo a dramatic 24-fold volume change, while a moderate 2.3-fold volume variation is observed for more rigid PMAA–(poly(N-vinylpyrrolidone) (PMAA–PVPON)5 capsules when solution pH is varied between 7.4 and 4. Despite that both types of capsules exhibit discoid-to-oblate ellipsoid transitions, a 3-fold greater swelling in radial dimensions is found for one-component systems due to a greater degree of the circular face bulging. We also show that (PMAA–PVPON)5 discoidal capsules interact differently with J774A.1 macrophages, HMVEC endothelial cells, and 4T1 breast cancer cells. The discoidal capsules show 60% lower internalization as compared to spherical capsules. Finally, hydrogel capsules demonstrate a 2-fold decrease in size upon internalization. These capsules represent a unique example of elastic hydrogel discoids capable of pH-induced drastic and reversible variations in aspect ratios. Considering the RBC-mimicking shape, their dimensions, and their capability to undergo pH-triggered intracellular responses, the hydrogel capsules demonstrate considerable potential as novel carriers in shape-regulated transport and cellular uptake. PMID:24848786

A Comprehensive in vitro and in silico Analysis of Antibiotics that Activate PXR and Induce CYP3A4 in Liver and Intestine

PubMed Central

Yasuda, Kazuto; Ranade, Aarati; Venkataramanan, Raman; Strom, Stephen; Chupka, Jonathan; Ekins, Sean; Schuetz, Erin; Bachmann, Kenneth

2015-01-01

We have investigated several in silico and in vitro methods in order to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate PXR and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative PCR, immunoblotting and testosterone 6β-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (>10-fold) by rifampin and troleandomycin; moderately (> 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin and (> 4-fold) by erythromycin; weakly (>2.4-fold) by nafcillin, cefaclor and sulfisoxazole; and (>2-fold) by cefadroxil and penicillin V. Similar though not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation. PMID:18505790

Evaluation of the time-concentration-mortality responses of Plutella xylostella larvae to the interaction of Isaria fumosorosea with the insecticides beta-cypermethrin and Bacillus thuringiensis.

PubMed

Nian, Xiao-ge; He, Yu-rong; Lu, Li-hua; Zhao, Rui

2015-02-01

Entomopathogenic fungi are potential candidates for controlling Plutella xylostella, a cosmopolitan pest of crucifers. In this study, bioassays were conducted to evaluate the interaction between Isaria fumosorosea and sublethal doses of two insecticides, beta-cypermethrin and Bacillus thuringiensis, against P. xylostella. Data of each assay were in good agreement with the time-concentration-mortality model, indicating a strong dependence of the fungus and insecticide interaction on both concentration and post-exposure time. Using beta-cypermethrin 58-116 µg mL(-1) or B. thuringiensis 222.5-890 µg mL(-1) with the fungus significantly enhanced fungal efficacy. The LC50 values of the fungus declined over a 1-7 day period after exposure, and the LT50 values decreased with increasing concentration. Based on LC50 or LC90 estimates, synergism between the fungus and beta-cypermethrin resulted in a 2.7-28.3-fold reduction in LC50 values and a 12.1-19.6-fold reduction in LC90 values, while synergism of the fungus with B. thuringiensis led to a 2.4-385.0-fold reduction in LC50 values and a 4.4-151.7-fold reduction in LC90 values. Results show that sublethal doses of B. thuringiensis and beta-cypermethrin can synergise I. fumosorosea activity on P. xylostella, suggesting that combination of I. fumosorosea with the two insecticides might offer an integrated approach to controlling P. xylostella in practice. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

Structural perturbations on huntingtin N17 domain during its folding on 2D-nanomaterials

NASA Astrophysics Data System (ADS)

Zhang, Leili; Feng, Mei; Zhou, Ruhong; Luan, Binquan

2017-09-01

A globular protein’s folded structure in its physiological environment is largely determined by its amino acid sequence. Recently, newly discovered transformer proteins as well as intrinsically disordered proteins may adopt the folding-upon-binding mechanism where their secondary structures are highly dependent on their binding partners. Due to the various applications of nanomaterials in biological sensors and potential wearable devices, it is important to discover possible conformational changes of proteins on nanomaterials. Here, through molecular dynamics simulations, we show that the first 17 residues of the huntingtin protein (HTT-N17) exhibit appreciable differences during its folding on 2D-nanomaterials, such as graphene and MoS2 nanosheets. Namely, the protein is disordered on the graphene surface but is helical on the MoS2 surface. Despite that the amphiphilic environment at the nanosheet-water interface promotes the folding of the amphipathic proteins (such as HTT-N17), competitions between protein-nanosheet and intra-protein interactions yield very different protein conformations. Therefore, as engineered binding partners, nanomaterials might significantly affect the structures of adsorbed proteins.

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

PubMed Central

Mustoe, Anthony M.; Al-Hashimi, Hashim M.; Brooks, Charles L.

2016-01-01

A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding. PMID:26481360

Screening and identification of lncRNAs as potential biomarkers for pulmonary tuberculosis.

PubMed

Chen, Zhong-Liang; Wei, Li-Liang; Shi, Li-Ying; Li, Meng; Jiang, Ting-Ting; Chen, Jing; Liu, Chang-Ming; Yang, Su; Tu, Hui-Hui; Hu, Yu-Ting; Gan, Lin; Mao, Lian-Gen; Wang, Chong; Li, Ji-Cheng

2017-12-01

Pulmonary tuberculosis (TB) is among the diseases with the highest morbidity and mortality worldwide. Effective diagnostic methods for TB are lacking. In this study, we investigated long non-coding RNAs (lncRNAs) in plasma using microarray and the potential diagnostic value of lncRNAs for TB. We found a total of 163 up-regulated lncRNAs and 348 down-regulated lncRNAs. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and coding-noncoding co-expression (CNC) analyses showed that functions of differentially expressed lncRNAs were mainly enriched in the regulation of alpha-beta T cell activation and the T cell receptor signalling pathway. Four differentially expressed lncRNAs, NR_038221 (fold change = 3.79, P < 0.01), NR_003142 (fold change = 1.69, P < 0.05), ENST00000570366 (fold change = 3.04, P < 0.05), and ENST00000422183 (fold change = 2.11, P < 0.001), were verified using RT-qPCR. Among those, NR_038221, NR_003142, and ENST00000570366 were found to be up-regulated, while ENST00000422183 was down-regulated. The value of the area under the curve (AUC) for the diagnostic model consisting of the four lncRNAs was 0.845 (sensitivity = 79.2%, specificity = 75%). We further predicted 85 mRNAs and 404 miRNAs that potentially interact with these lncRNAs. Our study revealed the potential value of lncRNAs as biomarkers for early diagnosis of TB and the underlying mechanisms of these abnormally expressed lncRNAs in the pathogenesis of TB.

All-atom four-body knowledge-based statistical potential to distinguish native tertiary RNA structures from nonnative folds.

PubMed

Masso, Majid

2018-09-14

Scientific breakthroughs in recent decades have uncovered the capability of RNA molecules to fulfill a wide array of structural, functional, and regulatory roles in living cells, leading to a concomitantly significant increase in both the number and diversity of experimentally determined RNA three-dimensional (3D) structures. Atomic coordinates from a representative training set of solved RNA structures, displaying low sequence and structure similarity, facilitate derivation of knowledge-based energy functions. Here we develop an all-atom four-body statistical potential and evaluate its capacity to distinguish native RNA 3D structures from nonnative folds based on calculated free energy scores. Atomic four-body nearest-neighbors are objectively identified by their occurrence as tetrahedral vertices in the Delaunay tessellations of RNA structures, and rates of atomic quadruplet interactions expected by chance are obtained from a multinomial reference distribution. Our four-body energy function, referred to as RAMP (ribonucleic acids multibody potential), is subsequently derived by applying the inverted Boltzmann principle to the frequency data, yielding an energy score for each type of atomic quadruplet interaction. Several well-known benchmark datasets reveal that RAMP is comparable with, and often outperforms, existing knowledge- and physics-based energy functions. To the best of our knowledge, this is the first study detailing an RNA tertiary structure-based multibody statistical potential and its comparative evaluation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Two-state protein model with water interactions: Influence of temperature on the intrinsic viscosity of myoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakk, Audun

2001-06-01

We describe a single-domain protein as a two-state system with water interactions. Around the unfolded apolar parts of the protein we incorporate the hydration effect by introducing hydrogen bonds between the water molecules in order to mimic the {open_quotes}icelike{close_quotes} shell structure. Intrinsic viscosity, proportional to the effective hydrodynamic volume, for sperm whale metmyoglobin is assigned from experimental data in the folded and in the denaturated state. By weighing statistically the two states against the degree of folding, we express the total intrinsic viscosity. The temperature dependence of the intrinsic viscosity, for different chemical potentials, is in good correspondence with experimentalmore » data [P. L. Privalov , J. Mol. Biol. >190, 487 (1986)]. Cold and warm unfolding, common to small globular proteins, is also a result of the model.« less

Shaping up the protein folding funnel by local interaction: lesson from a structure prediction study.

PubMed

Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

2006-02-28

Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of "chimera proteins." In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape.

Shaping up the protein folding funnel by local interaction: Lesson from a structure prediction study

PubMed Central

Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

2006-01-01

Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of “chimera proteins.” In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape. PMID:16488978

DNA Origami: Folded DNA-Nanodevices That Can Direct and Interpret Cell Behavior

PubMed Central

Kearney, Cathal J.; Lucas, Christopher R.; O'Brien, Fergal J.; Castro, Carlos E.

2016-01-01

DNA origami is a DNA-based nanotechnology that utilizes programmed combinations of short complementary oligonucleotides to fold a large single strand of DNA into precise 2-D and 3-D shapes. The exquisite nanoscale shape control of this inherently biocompatible material is combined with the potential to spatially address the origami structures with diverse cargos including drugs, antibodies, nucleic acid sequences, small molecules and inorganic particles. This programmable flexibility enables the fabrication of precise nanoscale devices that have already shown great potential for biomedical applications such as: drug delivery, biosensing and synthetic nanopore formation. In this Progress Report, we will review the advances in the DNA origami field since its inception several years ago and then focus on how these DNA-nanodevices can be designed to interact with cells to direct or probe their behavior. PMID:26840503

Forces Driving Chaperone Action

PubMed Central

Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C. A.

2016-01-01

SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188

Protein folding by NMR.

PubMed

Zhuravleva, Anastasia; Korzhnev, Dmitry M

2017-05-01

Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of desolvation barriers and sidechains on local-nonlocal coupling and chevron behaviors in coarse-grained models of protein folding.

PubMed

Chen, Tao; Chan, Hue Sun

2014-04-14

Local-nonlocal coupling is an organizational principle in protein folding. It envisions a cooperative energetic interplay between local conformational preferences and favorable nonlocal contacts. Previous theoretical studies by our group showed that two classes of native-centric coarse-grained models can capture the experimentally observed high degrees of protein folding cooperativity and diversity in folding rates. These models either embody an explicit local-nonlocal coupling mechanism or incorporate desolvation barriers in the models' pairwise interactions. Here a conceptual connection is made between these two paradigmatic coarse-grained interaction schemes by showing that desolvation barriers enhance local-nonlocal coupling. Furthermore, we find that a class of coarse-grained protein models with a single-site representation of sidechains also increases local-nonlocal coupling relative to mainchain models without sidechains. Enhanced local-nonlocal coupling generally leads to higher folding cooperativity and chevron plots with more linear folding arms. For the sidechain models studied, the chevron plot simulated with entirely native-centric intrachain interactions behaves very similarly to the corresponding chevron plots simulated with interactions that are partly modulated by sequence- and denaturant-dependent transfer free energies. In these essentially native-centric models, the mild chevron rollovers in the simulated folding arm are caused by occasionally populated intermediates as well as the movement of the unfolded and putative folding transition states. The strength and limitation of the models are analyzed by comparison with experiment. New formulations of sidechain models that may provide a physical account for nonnative interactions are also explored.

Direct protein photoinduced conformational changes using porphyrins.

NASA Astrophysics Data System (ADS)

Brancaleon, Lorenzo; Silva, Ivan; Fernandez, Nicholas; Johnson, Eric; Sansone, Samuel

2008-03-01

Most proteins functions depend on the interaction with other ligands. These interactions depend on uniquely structured binding sites formed by the folding of the proteins. Ligands can often prompt intended as well as ``accidental'' protein structural changes. One can foresee that the ability to prompt and control post-translational protein folding could be a powerful tool to investigate protein folding mechanisms but also to inhibit certain proteins or induce new properties to proteins. One possible way to produce such structural disruption is the combination of light and photoactive ligands. This option has been investigated in recent years by exploiting photoisomerization and other properties of non-physiological dyes. We used an alternative approach which uses porphyrins as the ``triggers'' of structural changes. The advantage of porphyrins is that they can be found naturally in living cells. The photophysical properties of porphyrins can induce local as well as long range effects on the structure of the bound protein. Porphyrins are known to produce structural changes in porphyrin-specific proteins, however the novelty of our results is that we demonstrated that these dyes can also produce structural changes in non-porphyrin-specific globular proteins. We will present an overview of our research to-date in this field and its potential applications.

Fold independent structural comparisons of protein-ligand binding sites for exploring functional relationships.

PubMed

Gold, Nicola D; Jackson, Richard M

2006-02-03

The rapid growth in protein structural data and the emergence of structural genomics projects have increased the need for automatic structure analysis and tools for function prediction. Small molecule recognition is critical to the function of many proteins; therefore, determination of ligand binding site similarity is important for understanding ligand interactions and may allow their functional classification. Here, we present a binding sites database (SitesBase) that given a known protein-ligand binding site allows rapid retrieval of other binding sites with similar structure independent of overall sequence or fold similarity. However, each match is also annotated with sequence similarity and fold information to aid interpretation of structure and functional similarity. Similarity in ligand binding sites can indicate common binding modes and recognition of similar molecules, allowing potential inference of function for an uncharacterised protein or providing additional evidence of common function where sequence or fold similarity is already known. Alternatively, the resource can provide valuable information for detailed studies of molecular recognition including structure-based ligand design and in understanding ligand cross-reactivity. Here, we show examples of atomic similarity between superfamily or more distant fold relatives as well as between seemingly unrelated proteins. Assignment of unclassified proteins to structural superfamiles is also undertaken and in most cases substantiates assignments made using sequence similarity. Correct assignment is also possible where sequence similarity fails to find significant matches, illustrating the potential use of binding site comparisons for newly determined proteins.

Impact of hydrodynamic interactions on protein folding rates depends on temperature

NASA Astrophysics Data System (ADS)

Zegarra, Fabio C.; Homouz, Dirar; Eliaz, Yossi; Gasic, Andrei G.; Cheung, Margaret S.

2018-03-01

We investigated the impact of hydrodynamic interactions (HI) on protein folding using a coarse-grained model. The extent of the impact of hydrodynamic interactions, whether it accelerates, retards, or has no effect on protein folding, has been controversial. Together with a theoretical framework of the energy landscape theory (ELT) for protein folding that describes the dynamics of the collective motion with a single reaction coordinate across a folding barrier, we compared the kinetic effects of HI on the folding rates of two protein models that use a chain of single beads with distinctive topologies: a 64-residue α /β chymotrypsin inhibitor 2 (CI2) protein, and a 57-residue β -barrel α -spectrin Src-homology 3 domain (SH3) protein. When comparing the protein folding kinetics simulated with Brownian dynamics in the presence of HI to that in the absence of HI, we find that the effect of HI on protein folding appears to have a "crossover" behavior about the folding temperature. This means that at a temperature greater than the folding temperature, the enhanced friction from the hydrodynamic solvents between the beads in an unfolded configuration results in lowered folding rate; conversely, at a temperature lower than the folding temperature, HI accelerates folding by the backflow of solvent toward the folded configuration of a protein. Additionally, the extent of acceleration depends on the topology of a protein: for a protein like CI2, where its folding nucleus is rather diffuse in a transition state, HI channels the formation of contacts by favoring a major folding pathway in a complex free energy landscape, thus accelerating folding. For a protein like SH3, where its folding nucleus is already specific and less diffuse, HI matters less at a temperature lower than the folding temperature. Our findings provide further theoretical insight to protein folding kinetic experiments and simulations.

Energetics of protein-DNA interactions.

PubMed

Donald, Jason E; Chen, William W; Shakhnovich, Eugene I

2007-01-01

Protein-DNA interactions are vital for many processes in living cells, especially transcriptional regulation and DNA modification. To further our understanding of these important processes on the microscopic level, it is necessary that theoretical models describe the macromolecular interaction energetics accurately. While several methods have been proposed, there has not been a careful comparison of how well the different methods are able to predict biologically important quantities such as the correct DNA binding sequence, total binding free energy and free energy changes caused by DNA mutation. In addition to carrying out the comparison, we present two important theoretical models developed initially in protein folding that have not yet been tried on protein-DNA interactions. In the process, we find that the results of these knowledge-based potentials show a strong dependence on the interaction distance and the derivation method. Finally, we present a knowledge-based potential that gives comparable or superior results to the best of the other methods, including the molecular mechanics force field AMBER99.

DETECTION OF TWO ISOMERIC BINDING CONFIGURATIONS IN A PROTEIN-APTAMER COMPLEX WITH A BIOLOGICAL NANOPORE

PubMed Central

Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni

2015-01-01

Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we have shown that folded proteins can be internalised inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalisation of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement, and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores might provide a new approach to probe conformational heterogeneity in protein:DNA interactions. PMID:25493908

Ibrutinib Dosing Strategies Based on Interaction Potential of CYP3A4 Perpetrators Using Physiologically Based Pharmacokinetic Modeling.

PubMed

de Zwart, L; Snoeys, J; De Jong, J; Sukbuntherng, J; Mannaert, E; Monshouwer, M

2016-11-01

Based on ibrutinib pharmacokinetics and potential sensitivity towards CYP3A4-mediated drug-drug interactions (DDIs), a physiologically based pharmacokinetic approach was developed to mechanistically describe DDI with various CYP3A4 perpetrators in healthy men under fasting conditions. These models were verified using clinical data for ketoconazole (strong CYP3A4 inhibitor) and used to prospectively predict and confirm the inducing effect of rifampin (strong CYP3A4 inducer); DDIs with mild (fluvoxamine, azithromycin) and moderate inhibitors (diltiazem, voriconazole, clarithromycin, itraconazole, erythromycin), and moderate (efavirenz) and strong CYP3A4 inducers (carbamazepine), were also predicted. Ketoconazole increased ibrutinib area under the curve (AUC) by 24-fold, while rifampin decreased ibrutinib AUC by 10-fold; coadministration of ibrutinib with strong inhibitors or inducers should be avoided. The ibrutinib dose should be reduced to 140 mg (quarter of maximal prescribed dose) when coadministered with moderate CYP3A4 inhibitors so that exposures remain within observed ranges at therapeutic doses. Thus, dose recommendations for CYP3A4 perpetrator use during ibrutinib treatment were developed and approved for labeling. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Asymmetric scoring functions for proteins

NASA Astrophysics Data System (ADS)

Lezon, Timothy; Holter, Neal; Maritan, Amos; Banavar, Jayanth

2003-03-01

The protein folding problem entails the prediction of the native state structure of a protein given the sequence of amino acids. In a coarse-grained description of a protein, an important ingredient for attempting this task is the determination of the effective energies of interaction between amino acids. We will discuss a simple approach for determining such interaction potentials from a training set of protein sequences and their experimentally determined native state structures. The key new ingredient in our study is the incorporation of the lack of symmetry in the effective interactions between amino acids. Our results, obtained using a set of 513 proteins, and their implications will be discussed.

Competition between protein folding and aggregation: A three-dimensional lattice-model simulation

NASA Astrophysics Data System (ADS)

Bratko, D.; Blanch, H. W.

2001-01-01

Aggregation of protein molecules resulting in the loss of biological activity and the formation of insoluble deposits represents a serious problem for the biotechnology and pharmaceutical industries and in medicine. Considerable experimental and theoretical efforts are being made in order to improve our understanding of, and ability to control, the process. In the present work, we describe a Monte Carlo study of a multichain system of coarse-grained model proteins akin to lattice models developed for simulations of protein folding. The model is designed to examine the competition between intramolecular interactions leading to the native protein structure, and intermolecular association, resulting in the formation of aggregates of misfolded chains. Interactions between the segments are described by a variation of the Go potential [N. Go and H. Abe, Biopolymers 20, 1013 (1981)] that extends the recognition between attracting types of segments to pairs on distinct chains. For the particular model we adopt, the global free energy minimum of a pair of protein molecules corresponds to a dimer of native proteins. When three or more molecules interact, clusters of misfolded chains can be more stable than aggregates of native folds. A considerable fraction of native structure, however, is preserved in these cases. Rates of conformational changes rapidly decrease with the size of the protein cluster. Within the timescale accessible to computer simulations, the folding-aggregation balance is strongly affected by kinetic considerations. Both the native form and aggregates can persist in metastable states, even if conditions such as temperature or concentration favor a transition to an alternative form. Refolding yield can be affected by the presence of an additional polymer species mimicking the function of a molecular chaperone.

Numerical Simulation of the Self-Oscillations of the Vocal Folds and of the Resulting Acoustic Phenomena in the Vocal Tract

NASA Astrophysics Data System (ADS)

Švancara, P.; Horáček, J.; Švec, J. G.

The study presents a three-dimensional (3D) finite element (FE) model of the flow-induced self-oscillation of the human vocal folds in interaction with acoustics of simplified vocal tract models. The 3D vocal tract models of the acoustic spaces shaped for simulation of phonation of Czech vowels [a:], [i:] and [u:] were created by converting the data from the magnetic resonance images (MRI). For modelling of the fluid-structure interaction, explicit coupling scheme with separated solvers for fluid and structure domain was utilized. The FE model comprises vocal folds pretension before starting phonation, large deformations of the vocal fold tissue, vocal-fold collisions, fluid-structure interaction, morphing the fluid mesh according to the vocal-fold motion (Arbitrary Lagrangian-Eulerian approach), unsteady viscous compressible airflow described by the Navier-Stokes equations and airflow separation. The developed FE model enables to study the relationship between flow-induced vibrations of the vocal folds and acoustic wave propagation in the vocal tract and can also be used to simulate for example pathological changes in the vocal fold tissue and their influence on the voice production.

Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

PubMed

Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

2016-01-01

While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

Native Contact Density and Nonnative Hydrophobic Effects in the Folding of Bacterial Immunity Proteins

PubMed Central

Chen, Tao; Chan, Hue Sun

2015-01-01

The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate. PMID:26016652

Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM.

PubMed

Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie; Lahtinen, Sampo J; Brix, Susanne; Abou Hachem, Maher; Svensson, Birte

2017-06-23

Adhesion to intestinal mucosa is a crucial property for probiotic bacteria. Adhesion is thought to increase host-bacterial interactions, thus potentially enabling health benefits to the host. Molecular events connected with adhesion and surface proteome changes were investigated for the probiotic Lactobacillus acidophilus NCFM cultured with established or emerging prebiotic carbohydrates as carbon source and in the presence of mucin, the glycoprotein of the epithelial mucus layer. Variation in adhesion to HT29-cells and mucin was associated with carbon source and mucin-induced subproteome abundancy differences. Specifically, while growth on fructooligosaccharides (FOS) only stimulated adhesion to intestinal HT-29 cells, cellobiose and polydextrose in addition increased adhesion to mucin. Adhesion to HT-29 cells increased by about 2-fold for bacteria grown on mucin-supplemented glucose. Comparative 2DE-MS surface proteome analysis showed different proteins in energy metabolism appearing on the surface, suggesting they exert moonlighting functions. Mucin-supplemented bacteria had relative abundance of pyruvate kinase and fructose-bisphosphate aldolase increased by about 2-fold while six spots with 3.2-2.1 fold reduced relative abundance comprised elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, and manganese-dependent inorganic pyrophosphatase. Surface proteome of cellobiose- compared to glucose-grown L. acidophilus NCFM had phosphate starvation inducible protein stress-related, thermostable pullulanase, and elongation factor G increasing 4.4-2.4 fold, while GAPDH, elongation factor Ts, and pyruvate kinase were reduced by 2.0-1.5 fold in relative abundance. Addition of recombinant L. acidophilus NCFM elongation factor G and pyruvate kinase to a coated mucin layer significantly suppressed subsequent adhesion of the bacterium. Human diet is important for intestinal health and food components, especially non-digestible carbohydrates can beneficially modify the microbiota. In the present study, effects of emerging and established prebiotic carbohydrates on the probiotic potential of Lactobacillus acidophilus NCFM were investigated by testing adhesion to a mucin layer and intestinal cells, and comparing this with changes in abundancy of surface proteins thought to be important for host interactions. Increased adhesion was observed following culturing of the bacterium with fructooligosaccharides, cellobiose or polydextrose, as well as mucin-supplemented glucose as carbon source. Enhanced adhesion ability can prolong bacterial residence in GIT yielding positive health effects. Higher relative abundance of certain surface proteins under various conditions (i.e. grown on cellobiose or mucin-supplemented glucose) suggested involvement of these proteins in adhesion, as confirmed by competition in case of two recombinantly produced moonlighting proteins. Combination of Lactobacillus acidophilus NCFM with different carbohydrates revealed potential bacterial determinants of synbiotic interactions, including stimulation of adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

Complexes of native Ubiquitin and dodecyl sulfate illustrate the nature of hydrophobic and electrostatic interactions in the binding of proteins and surfactants

PubMed Central

Shaw, Bryan F.; Schneider, Grégory F.; Arthanari, Haribabu; Narovlyansky, Max; Moustakas, Demetri; Durazo, Armando; Wagner, Gerhard; Whitesides, George M.

2011-01-01

A previous study, using capillary electrophoresis (CE), reported that six discrete complexes of ubiquitin (UBI) and sodium dodecyl sulfate (SDS) form at different concentrations of SDS along the pathway to unfolding of UBI in solutions of SDS. One complex (which formed between 0.8 and 1.8 mM SDS) consisted of native UBI associated with approximately 11 molecules of SDS. The current study used CE and 15N/13C-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy to identify residues in folded UBI that associate specifically with SDS at 0.8-1.8 mM SDS, and to correlate these associations with established biophysical and structural properties of this well-characterized protein. The ability of the surface charge and hydrophobicity of folded UBI to affect the association with SDS (at concentrations below the CMC) was studied, using CE, by converting lys-ε-NH3+ to lys-ε-NHCOCH3 groups. According to CE, the acetylation of lysine residues inhibited the binding of 11 SDS ([SDS] < 2 mM) and decreased the number of complexes of composition UBI-(NHAc)8·SDSn that formed on the pathway of unfolding of UBI-(NHAc)8 in SDS. A comparison of 15N-1H HSQC spectra at 0 mM and 1 mM SDS with calculated electrostatic surface potentials of folded UBI (e.g., solutions to the non-linear Poisson-Boltzmann (PB) equation) suggested, however, that SDS binds preferentially to native UBI at hydrophobic residues that are formally neutral (i.e., Leu and Ile), but that have positive electrostatic surface potential (as predicted from solutions to non-linear Poisson-Boltzmann equations); SDS did not uniformly interact with residues that have formal positive charge (e.g., Lys or Arg). Cationic functional groups, therefore, promote the binding of SDS to folded UBI because these groups exert long-range effects on the positive electrostatic surface potential (which extend beyond their own van der Waal’s radii, as predicted from PB theory), and not because cationic groups are necessarily the site of ionic interactions with sulfate groups. Moreover, SDS associated with residues in native UBI without regard to their location in α-helix or β-sheet structure (although residues in hydrogen-bonded loops did not bind SDS). No correlation was observed between the association of an amino acid with SDS and the solvent accessibility of the residue or its rate of amide H/D exchange. This study establishes a few (of perhaps several) factors that control the simultaneous molecular recognition of multiple anionic amphiphiles by a folded cytosolic protein. PMID:21939262

Predicting Electrostatic Forces in RNA Folding

PubMed Central

Tan, Zhi-Jie; Chen, Shi-Jie

2016-01-01

Metal ion-mediated electrostatic interactions are critical to RNA folding. Although considerable progress has been made in mechanistic studies, the problem of accurate predictions for the ion effects in RNA folding remains unsolved, mainly due to the complexity of several potentially important issues such as ion correlation and dehydration effects. In this chapter, after giving a brief overview of the experimental findings and theoretical approaches, we focus on a recently developed new model, the tightly bound ion (TBI) model, for ion electrostatics in RNA folding. The model is unique because it can treat ion correlation and fluctuation effects for realistic RNA 3D structures. For monovalent ion (such as Na+) solutions, where ion correlation is weak, TBI and the Poisson–Boltzmann (PB) theory give the same results and the results agree with the experimental data. For multivalent ion (such as Mg2+) solutions, where ion correlation can be strong, however, TBI gives much improved predictions than the PB. Moreover, the model suggests an ion correlation- induced mechanism for the unusual efficiency of Mg2+ ions in the stabilization of RNA tertiary folds. In this chapter, after introducing the theoretical framework of the TBI model, we will describe how to apply the model to predict ion-binding properties and ion-dependent folding stabilities. PMID:20946803

ROTAS: a rotamer-dependent, atomic statistical potential for assessment and prediction of protein structures.

PubMed

Park, Jungkap; Saitou, Kazuhiro

2014-09-18

Multibody potentials accounting for cooperative effects of molecular interactions have shown better accuracy than typical pairwise potentials. The main challenge in the development of such potentials is to find relevant structural features that characterize the tightly folded proteins. Also, the side-chains of residues adopt several specific, staggered conformations, known as rotamers within protein structures. Different molecular conformations result in different dipole moments and induce charge reorientations. However, until now modeling of the rotameric state of residues had not been incorporated into the development of multibody potentials for modeling non-bonded interactions in protein structures. In this study, we develop a new multibody statistical potential which can account for the influence of rotameric states on the specificity of atomic interactions. In this potential, named "rotamer-dependent atomic statistical potential" (ROTAS), the interaction between two atoms is specified by not only the distance and relative orientation but also by two state parameters concerning the rotameric state of the residues to which the interacting atoms belong. It was clearly found that the rotameric state is correlated to the specificity of atomic interactions. Such rotamer-dependencies are not limited to specific type or certain range of interactions. The performance of ROTAS was tested using 13 sets of decoys and was compared to those of existing atomic-level statistical potentials which incorporate orientation-dependent energy terms. The results show that ROTAS performs better than other competing potentials not only in native structure recognition, but also in best model selection and correlation coefficients between energy and model quality. A new multibody statistical potential, ROTAS accounting for the influence of rotameric states on the specificity of atomic interactions was developed and tested on decoy sets. The results show that ROTAS has improved ability to recognize native structure from decoy models compared to other potentials. The effectiveness of ROTAS may provide insightful information for the development of many applications which require accurate side-chain modeling such as protein design, mutation analysis, and docking simulation.

Prelude and Fugue, predicting local protein structure, early folding regions and structural weaknesses.

PubMed

Kwasigroch, Jean Marc; Rooman, Marianne

2006-07-15

Prelude&Fugue are bioinformatics tools aiming at predicting the local 3D structure of a protein from its amino acid sequence in terms of seven backbone torsion angle domains, using database-derived potentials. Prelude(&Fugue) computes all lowest free energy conformations of a protein or protein region, ranked by increasing energy, and possibly satisfying some interresidue distance constraints specified by the user. (Prelude&)Fugue detects sequence regions whose predicted structure is significantly preferred relative to other conformations in the absence of tertiary interactions. These programs can be used for predicting secondary structure, tertiary structure of short peptides, flickering early folding sequences and peptides that adopt a preferred conformation in solution. They can also be used for detecting structural weaknesses, i.e. sequence regions that are not optimal with respect to the tertiary fold. http://babylone.ulb.ac.be/Prelude_and_Fugue.

Synthetic oligorotaxanes exert high forces when folding under mechanical load

NASA Astrophysics Data System (ADS)

Sluysmans, Damien; Hubert, Sandrine; Bruns, Carson J.; Zhu, Zhixue; Stoddart, J. Fraser; Duwez, Anne-Sophie

2018-01-01

Folding is a ubiquitous process that nature uses to control the conformations of its molecular machines, allowing them to perform chemical and mechanical tasks. Over the years, chemists have synthesized foldamers that adopt well-defined and stable folded architectures, mimicking the control expressed by natural systems1,2. Mechanically interlocked molecules, such as rotaxanes and catenanes, are prototypical molecular machines that enable the controlled movement and positioning of their component parts3-5. Recently, combining the exquisite complexity of these two classes of molecules, donor-acceptor oligorotaxane foldamers have been synthesized, in which interactions between the mechanically interlocked component parts dictate the single-molecule assembly into a folded secondary structure6-8. Here we report on the mechanochemical properties of these molecules. We use atomic force microscopy-based single-molecule force spectroscopy to mechanically unfold oligorotaxanes, made of oligomeric dumbbells incorporating 1,5-dioxynaphthalene units encircled by cyclobis(paraquat-p-phenylene) rings. Real-time capture of fluctuations between unfolded and folded states reveals that the molecules exert forces of up to 50 pN against a mechanical load of up to 150 pN, and displays transition times of less than 10 μs. While the folding is at least as fast as that observed in proteins, it is remarkably more robust, thanks to the mechanically interlocked structure. Our results show that synthetic oligorotaxanes have the potential to exceed the performance of natural folding proteins.

The H2A-H2B dimeric kinetic intermediate is stabilized by widespread hydrophobic burial with few fully native interactions.

PubMed

Guyett, Paul J; Gloss, Lisa M

2012-01-20

The H2A-H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within ∼5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I₂ and I₂*, the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I₂ ensemble folds to the native heterodimer, N₂, through an observable, first-order kinetic phase. To determine the regions of structure in the I₂ ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I₂, the transition state and N₂; however, only residues in the hydrophobic core of the dimer interface perturbed the I₂* population. Destabilization of I₂* correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Φ values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I₂ and N₂, but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I₂. These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I₂, but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N₂, more native-like interactions are developed and nonnative interactions are rearranged. Copyright © 2011 Elsevier Ltd. All rights reserved.

An Immersed-Boundary Method for Fluid-Structure Interaction in the Human Larynx

NASA Astrophysics Data System (ADS)

Luo, Haoxiang; Zheng, Xudong; Mittal, Rajat; Bielamowicz, Steven

2006-11-01

We describe a novel and accurate computational methodology for modeling the airflow and vocal fold dynamics in human larynx. The model is useful in helping us gain deeper insight into the complicated bio-physics of phonation, and may have potential clinical application in design and placement of synthetic implant in vocal fold surgery. The numerical solution of the airflow employs a previously developed immersed-boundary solver. However, in order to incorporate the vocal fold into the model, we have developed a new immersed-boundary method that can simulate the dynamics of the multi-layered, viscoelastic solids. In this method, a finite-difference scheme is used to approximate the derivatives and ghost cells are defined near the boundary. To impose the traction boundary condition, a third-order polynomial is obtained using the weighted least squares fitting to approximate the function locally. Like its analogue for the flow solver, this immersed-boundary method for the solids has the advantage of simple grid generation, and may be easily implemented on parallel computers. In the talk, we will present the simulation results on both the specified vocal fold motion and the flow-induced vocal fold vibration. Supported by NIDCD Grant R01 DC007125-01A1.

Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape.

PubMed

Adamcik, Jozef; Mezzenga, Raffaele

2018-02-15

Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on- or off-pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon↛crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analyzing the effect of homogeneous frustration in protein folding.

PubMed

Contessoto, Vinícius G; Lima, Debora T; Oliveira, Ronaldo J; Bruni, Aline T; Chahine, Jorge; Leite, Vitor B P

2013-10-01

The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization, and functional transitions. According to the theory, the energy landscape of protein folding is funneled toward the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution, satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of 19 proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well-separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of non-native contact interactions in different folding scenarios. These findings strongly correlate with the protein free-energy folding barrier and the absolute contact order parameters. These computational results are corroborated by principal component analysis and partial least square techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. Copyright © 2013 Wiley Periodicals, Inc.

Self-folding mechanics of graphene tearing and peeling from a substrate

NASA Astrophysics Data System (ADS)

He, Ze-Zhou; Zhu, Yin-Bo; Wu, Heng-An

2018-06-01

Understanding the underlying mechanism in the tearing and peeling processes of graphene is crucial for the further hierarchical design of origami-like folding and kirigami-like cutting of graphene. However, the complex effects among bending moduli, adhesion, interlayer interaction, and local crystal structure during origami-like folding and kirigami-like cutting remain unclear, resulting in challenges to the practical applications of existing theoretical and experimental findings as well as to potential manipulations of graphene in metamaterials and nanodevices. Toward this end, classical molecular dynamics (MD) simulations are performed with synergetic theoretical analysis to explore the tearing and peeling of self-folded graphene from a substrate driven by external force and by thermal activation. It is found that the elastic energy localized at the small folding ridge plays a significant role in the crack trajectory. Due to the extremely small bending modulus of monolayer graphene, its taper angle when pulled by an external force follows a scaling law distinct from that in case of bilayer graphene. With the increase in the initial width of the folding ridge, the self-folded graphene, motivated by thermal fluctuations, can be self-assembled by spontaneous self-tearing and peeling from a substrate. Simultaneously, the scaling law between the taper angle and adhesive energy is independent of the motivations for thermal activation-induced self-assembly and external force tearing, providing effective insights into the underlying physics for graphene-based origami-like folding and kirigami-like cutting.

Fully-coupled aeroelastic simulation with fluid compressibility — For application to vocal fold vibration

PubMed Central

Yang, Jubiao; Wang, Xingshi; Krane, Michael; Zhang, Lucy T.

2017-01-01

In this study, a fully-coupled fluid–structure interaction model is developed for studying dynamic interactions between compressible fluid and aeroelastic structures. The technique is built based on the modified Immersed Finite Element Method (mIFEM), a robust numerical technique to simulate fluid–structure interactions that has capabilities to simulate high Reynolds number flows and handles large density disparities between the fluid and the solid. For accurate assessment of this intricate dynamic process between compressible fluid, such as air and aeroelastic structures, we included in the model the fluid compressibility in an isentropic process and a solid contact model. The accuracy of the compressible fluid solver is verified by examining acoustic wave propagations in a closed and an open duct, respectively. The fully-coupled fluid–structure interaction model is then used to simulate and analyze vocal folds vibrations using compressible air interacting with vocal folds that are represented as layered viscoelastic structures. Using physiological geometric and parametric setup, we are able to obtain a self-sustained vocal fold vibration with a constant inflow pressure. Parametric studies are also performed to study the effects of lung pressure and vocal fold tissue stiffness in vocal folds vibrations. All the case studies produce expected airflow behavior and a sustained vibration, which provide verification and confidence in our future studies of realistic acoustical studies of the phonation process. PMID:29527067

Development, validation and utility of an in vitro technique for assessment of potential clinical drug-drug interactions involving P-glycoprotein.

PubMed

Keogh, John P; Kunta, Jeevan R

2006-04-01

Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug-drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug-transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [3H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC50) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [3H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A-->B] transport, [3H]-digoxin [B-->A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 microM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC50 values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC50 values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin-drug interactions mediated via Pgp.

Free Energy Landscape and Multiple Folding Pathways of an H-Type RNA Pseudoknot

PubMed Central

Bian, Yunqiang; Zhang, Jian; Wang, Jun; Wang, Jihua; Wang, Wei

2015-01-01

How RNA sequences fold to specific tertiary structures is one of the key problems for understanding their dynamics and functions. Here, we study the folding process of an H-type RNA pseudoknot by performing a large-scale all-atom MD simulation and bias-exchange metadynamics. The folding free energy landscapes are obtained and several folding intermediates are identified. It is suggested that the folding occurs via multiple mechanisms, including a step-wise mechanism starting either from the first helix or the second, and a cooperative mechanism with both helices forming simultaneously. Despite of the multiple mechanism nature, the ensemble folding kinetics estimated from a Markov state model is single-exponential. It is also found that the correlation between folding and binding of metal ions is significant, and the bound ions mediate long-range interactions in the intermediate structures. Non-native interactions are found to be dominant in the unfolded state and also present in some intermediates, possibly hinder the folding process of the RNA. PMID:26030098

Internal friction controls the speed of protein folding from a compact configuration.

PubMed

Pabit, Suzette A; Roder, Heinrich; Hagen, Stephen J

2004-10-05

Several studies have found millisecond protein folding reactions to be controlled by the viscosity of the solvent: Reducing the viscosity allows folding to accelerate. In the limit of very low solvent viscosity, however, one expects a different behavior. Internal interactions, occurring within the solvent-excluded interior of a compact molecule, should impose a solvent-independent upper limit to folding speed once the bulk diffusional motions become sufficiently rapid. Why has this not been observed? We have studied the effect of solvent viscosity on the folding of cytochrome c from a highly compact, late-stage intermediate configuration. Although the folding rate accelerates as the viscosity declines, it tends toward a finite limiting value approximately 10(5) s(-1) as the viscosity tends toward zero. This limiting rate is independent of the cosolutes used to adjust solvent friction. Therefore, interactions within the interior of a compact denatured polypeptide can limit the folding rate, but the limiting time scale is very fast. It is only observable when the solvent-controlled stages of folding are exceedingly rapid or else absent. Interestingly, we find a very strong temperature dependence in these "internal friction"-controlled dynamics, indicating a large energy scale for the interactions that govern reconfiguration within compact, near-native states of a protein.

Aromatic residues engineered into the beta-turn nucleation site of ubiquitin lead to a complex folding landscape, non-native side-chain interactions, and kinetic traps.

PubMed

Rea, Anita M; Simpson, Emma R; Meldrum, Jill K; Williams, Huw E L; Searle, Mark S

2008-12-02

The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.

Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gracia, Tannia; Hilscherova, Klara; Jones, Paul D.

2007-12-01

The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3{beta}HSD2, CYP11{beta}1, CYP11{beta}2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11{beta}2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantlymore » increased CYP11{beta}2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The {beta}-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different.« less

Molecular simulation of surfactant-assisted protein refolding

NASA Astrophysics Data System (ADS)

Lu, Diannan; Liu, Zheng; Liu, Zhixia; Zhang, Minlian; Ouyang, Pingkai

2005-04-01

Protein refolding to its native state in vitro is a challenging problem in biotechnology, i.e., in the biomedical, pharmaceutical, and food industry. Protein aggregation and misfolding usually inhibit the recovery of proteins with their native states. These problems can be partially solved by adding a surfactant into a suitable solution environment. However, the process of this surfactant-assisted protein refolding is not well understood. In this paper, we wish to report on the first-ever simulations of surfactant-assisted protein refolding. For these studies, we defined a simple model for the protein and the surfactant and investigated how a surfactant affected the folding behavior of a two-dimensional lattice protein molecule. The model protein and model surfactant were chosen such that we could capture the important features of the folding process and the interaction between the protein and the surfactant, namely, the hydrophobic interaction. It was shown that, in the absence of surfactants, a protein in an "energy trap" conformation, i.e., a local energy minima, could not fold into the native form, which was characterized by a global energy minimum. The addition of surfactants created folding pathways via the formation of protein-surfactant complexes and thus enabled the conformations that fell into energy trap states to escape from these traps and to form the native proteins. The simulation results also showed that it was necessary to match the hydrophobicity of surfactant to the concentration of denaturant, which was added to control the folding or unfolding of a protein. The surfactants with different hydrophobicity had their own concentration range on assisting protein refolding. All of these simulations agreed well with experimental results reported elsewhere, indicating both the validity of the simulations presented here and the potential application of the simulations for the design of a surfactant on assisting protein refolding.

How Well Does a Funneled Energy Landscape Capture the Folding Mechanism of Spectrin Domains?

PubMed Central

2013-01-01

Three structurally similar domains from α-spectrin have been shown to fold very differently. Firstly, there is a contrast in the folding mechanism, as probed by Φ-value analysis, between the R15 domain and the R16 and R17 domains. Secondly, there are very different contributions from internal friction to folding: the folding rate of the R15 domain was found to be inversely proportional to solvent viscosity, showing no apparent frictional contribution from the protein, but in the other two domains a large internal friction component was evident. Non-native misdocking of helices has been suggested to be responsible for this phenomenon. Here, I study the folding of these three proteins with minimalist coarse-grained models based on a funneled energy landscape. Remarkably, I find that, despite the absence of non-native interactions, the differences in folding mechanism of the domains are well captured by the model, and the agreement of the Φ-values with experiment is fairly good. On the other hand, within the context of this model, there are no significant differences in diffusion coefficient along the chosen folding coordinate, and the model cannot explain the large differences in folding rates between the proteins found experimentally. These results are nonetheless consistent with the expectations from the energy landscape perspective of protein folding: namely, that the folding mechanism is primarily determined by the native-like interactions present in the Gō-like model, with missing non-native interactions being required to explain the differences in “internal friction” seen in experiment. PMID:23947368

Structures and properties of molecular torsion balances to decipher the nature of substituent effects on the aromatic edge-to-face interaction.

PubMed

Gardarsson, Haraldur; Schweizer, W Bernd; Trapp, Nils; Diederich, François

2014-04-14

Various recent computational studies initiated this systematic re-investigation of substituent effects on aromatic edge-to-face interactions. Five series of Tröger base derived molecular torsion balances (MTBs), initially introduced by Wilcox and co-workers, showing an aromatic edge-to-face interaction in the folded, but not in the unfolded form, were synthesized. A fluorine atom or a trifluoromethyl group was introduced onto the edge ring in ortho-, meta-, and para-positions to the C-H group interacting with the face component. The substituents on the face component were varied from electron-donating to electron-withdrawing. Extensive X-ray crystallographic data allowed for a discussion on the conformational behavior of the torsional balances in the solid state. While most systems adopt the folded conformation, some were found to form supramolecular intercalative dimers, lacking the intramolecular edge-to-face interaction, which is compensated by the gain of aromatic π-stacking interactions between four aryl rings of the two molecular components. This dimerization does not take place in solution. The folding free enthalpy ΔG(fold) of all torsion balances was determined by (1)H NMR measurements by using 10 mM solutions of samples in CDCl3 and C6D6. Only the ΔG(fold) values of balances bearing an edge-ring substituent in ortho-position to the interacting C-H show a steep linear correlation with the Hammett parameter (σ(meta)) of the face-component substituent. Thermodynamic analysis using van't Hoff plots revealed that the interaction is enthalpy-driven. The ΔG(fold) values of the balances, in addition to partial charge calculations, suggest that increasing the polarization of the interacting C-H group makes a favorable contribution to the edge-to-face interaction. The largest contribution, however, seems to originate from local direct interactions between the substituent in ortho-position to the edge-ring C-H and the substituted face ring. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements

PubMed Central

PENEDO, J. CARLOS; WILSON, TIMOTHY J.; JAYASENA, SUMEDHA D.; KHVOROVA, ANASTASIA; LILLEY, DAVID M.J.

2004-01-01

It has been shown that the activity of the hammerhead ribozyme at μM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the μM range of magnesium ion concentration. Disruption of the loop–loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions. PMID:15100442

Synergistic Association of Genetic Variants with Environmental Risk Factors in Susceptibility to Essential Hypertension.

PubMed

Sousa, Ana Célia; Mendonça, Maria I; Pereira, Andreia; Gouveia, Sara; Freitas, Ana I; Guerra, Graça; Rodrigues, Mariana; Henriques, Eva; Freitas, Sónia; Borges, Sofia; Pereira, Décio; Brehm, António; Palma Dos Reis, Roberto

2017-10-01

Essential hypertension (EH) is a disease in which both environment and genes have an important role. This study was designed to identify the interaction model between genetic variants and environmental risk factors that most highly potentiates EH development. We performed a case-control study with 1641 participants (mean age 50.6 ± 8.1 years), specifically 848 patients with EH and 793 controls, adjusted for gender and age. Traditional risk factors, biochemical and genetic parameters, including the genotypic discrimination of 14 genetic variants previously associated with EH, were investigated. Multifactorial dimensionality reduction (MDR) software was used to analyze gene-environment interactions. Validation was performed using logistic regression analysis with environmental risk factors, significant genetic variants, and the best MDR model. The best model indicates that the interactions among the ADD1 rs4961 640T allele, diabetes, and obesity (body mass index ≥30) increase approximately four-fold the risk of EH (odds ratio = 3.725; 95% confidence interval: 2.945-4.711; p < 0.0001). This work showed that the interaction between the ADD1 rs4961 variant, obesity, and the presence of diabetes increased the susceptibility to EH four-fold. In these circumstances, lifestyle adjustment and diabetes control should be intensified in patients who carry the ADD1 variant.

Structural transitions in vortex systems with anisotropic interactions

DOE PAGES

Olszewski, Maciej W.; Eskildsen, M. R.; Reichhardt, Charles; ...

2017-12-29

We introduce a model of vortices in type-II superconductors with a four-fold anisotropy in the vortex–vortex interaction potential. Using numerical simulations we show that the vortex lattice undergoes structural transitions as the anisotropy is increased, with a triangular lattice at low anisotropy, a rhombic intermediate state, and a square lattice for high anisotropy. In some cases we observe a multi-q state consisting of an Archimedean tiling that combines square and triangular local ordering. At very high anisotropy, domains of vortex chain states appear. We discuss how this model can be generalized to higher order anisotropy as well as its applicabilitymore » to other particle-based systems with anisotropic particle–particle interactions.« less

Interactions of hyaluronan grafted on protein surfaces studied using a quartz crystal microbalance and a surface force balance.

PubMed

Jiang, Lei; Han, Juan; Yang, Limin; Ma, Hongchao; Huang, Bo

2015-10-07

Vocal folds are complex and multilayer-structured where the main layer is widely composed of hyaluronan (HA). The viscoelasticity of HA is key to voice production in the vocal fold as it affects the initiation and maintenance of phonation. In this study a simple layer-structured surface model was set up to mimic the structure of the vocal folds. The interactions between two opposing surfaces bearing HA were measured and characterised to analyse HA's response to the normal and shear compression at a stress level similar to that in the vocal fold. From the measurements of the quartz crystal microbalance, atomic force microscopy and the surface force balance, the osmotic pressure, normal interactions, elasticity change, volume fraction, refractive index and friction of both HA and the supporting protein layer were obtained. These findings may shed light on the physical mechanism of HA function in the vocal fold and the specific role of HA as an important component in the effective treatment of the vocal fold disease.

Green synthesis of zero-valent Fe-nanoparticles: Catalytic degradation of rhodamine B, interactions with bovine serum albumin and their enhanced antimicrobial activities.

PubMed

Khan, Zaheer; Al-Thabaiti, Shaeel Ahmad

2018-03-01

Biomimetic method was used for the synthesis of Fe-nanoparticles (FeNPs). FeCl 3 and Hibiscus sabdariffa, Roselle flower aqueous extract (HBS) were employed in the present studies. The FeNPs have been characterized by using UV-visible spectroscopy, transmission electron microscope (TEM), and energy dispersion X-ray spectroscopy (EDS). The average particles diameter was found to be 18 nm. The as prepared FeNPs were used as a catalyst to the oxidative degradation of rhodamine B (RB) in presence of NaBH 4 . The effects of various quencher on the degradation rates were examined by employing ammonium oxalate (AO), benzoquinone (BQ), isopropyl alcohol (IPA), and potassium iodide (KI). The interactions of FeNPs with bovine serum albumin (BSA) have been determined and discussed. Adsorption of FeNPs into the core of BSA changes the tryptophan environment from hydrophobic to hydrophilic (from folding to partially folded and/or unfolded). Tryptophan residues, indole moieties of BSA were responsible to complex formation with FeNPs in excited states via electrostatic, van der Waals, hydrogen bonding, hydrophobic and hydrophilic interactions with static quenching. The antimicrobial activities of FeNPs have been determined against human pathogens. Hibiscus sabdariffa flower extract shows mild antimicrobial activities against all target pathogenic organisms. FeNPs have potential antimicrobial activity against both bacterial strains and candida fungus even at low concentration, and retains potential application in biomedical industries. Copyright © 2018 Elsevier B.V. All rights reserved.

Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

PubMed Central

Bastolla, Ugo

2014-01-01

The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change. PMID:24970217

Surface-peaked medium effects in the interaction of nucleons with finite nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aguayo, F. J.; Arellano, H. F.

We investigate the asymptotic separation of the optical model potential for nucleon-nucleus scattering in momentum space, where the potential is split into a medium-independent term and another depending exclusively on the gradient of the density-dependent g matrix. This decomposition confines the medium sensitivity of the nucleon-nucleus coupling to the surface of the nucleus. We examine this feature in the context of proton-nucleus scattering at beam energies between 30 and 100 MeV and find that the pn coupling accounts for most of this sensitivity. Additionally, based on this general structure of the optical potential we are able to treat both, themore » medium dependence of the effective interaction and the full mixed density as described by single-particle shell models. The calculated scattering observables agree within 10% with those obtained by Arellano, Brieva, and Love in their momentum-space g-folding approach.« less

Ab initio RNA folding by discrete molecular dynamics: From structure prediction to folding mechanisms

PubMed Central

Ding, Feng; Sharma, Shantanu; Chalasani, Poornima; Demidov, Vadim V.; Broude, Natalia E.; Dokholyan, Nikolay V.

2008-01-01

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 Å deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNAPhe, pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses. PMID:18456842

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

PubMed Central

Smith, Everett Clinton; Culler, Megan R.; Hellman, Lance M.; Fried, Michael G.; Creamer, Trevor P.

2012-01-01

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

Discrete-to-continuum simulation approach to polymer chain systems: Subdiffusion, segregation, and chain folding

NASA Astrophysics Data System (ADS)

Foo, Grace M.; Pandey, R. B.

1998-05-01

A discrete-to-continuum approach is introduced to study the static and dynamic properties of polymer chain systems with a bead-spring chain model in two dimensions. A finitely extensible nonlinear elastic potential is used for the bond between the consecutive beads with the Lennard-Jones (LJ) potential with smaller (Rc=21/6σ=0.95) and larger (Rc=2.5σ=2.1) values of the upper cutoff for the nonbonding interaction among the neighboring beads. We find that chains segregate at temperature T=1.0 with Rc=2.1 and remain desegregated with Rc=0.95. At low temperature (T=0.2), chains become folded, in a ribbonlike conformation, unlike random and self-avoiding walk conformations at T=1.0. The power-law dependence of the rms displacements of the center of mass (Rc.m.) of the chains and their center node (Rcn) with time are nonuniversal, with the range of exponents ν1~=0.45-0.25 and ν2~=0.30-0.10, respectively. Both radius of gyration (Rg) and average bond length () decrease on increasing the range of interaction (Rc), consistent with the extended state in good solvent to collapsed state in poor solvent description of the polymer chains. Analysis of the radial distribution function supports these observations.

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

PubMed

Jia, Yan; Liu, Hui; Bao, Wei; Weng, Meizhi; Chen, Wei; Cai, Yongjun; Zheng, Zhongliang; Zou, Guolin

2010-12-01

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ab initio folding of proteins using all-atom discrete molecular dynamics

PubMed Central

Ding, Feng; Tsao, Douglas; Nie, Huifen; Dokholyan, Nikolay V.

2008-01-01

Summary Discrete molecular dynamics (DMD) is a rapid sampling method used in protein folding and aggregation studies. Until now, DMD was used to perform simulations of simplified protein models in conjunction with structure-based force fields. Here, we develop an all-atom protein model and a transferable force field featuring packing, solvation, and environment-dependent hydrogen bond interactions. Using the replica exchange method, we perform folding simulations of six small proteins (20–60 residues) with distinct native structures. In all cases, native or near-native states are reached in simulations. For three small proteins, multiple folding transitions are observed and the computationally-characterized thermodynamics are in quantitative agreement with experiments. The predictive power of all-atom DMD highlights the importance of environment-dependent hydrogen bond interactions in modeling protein folding. The developed approach can be used for accurate and rapid sampling of conformational spaces of proteins and protein-protein complexes, and applied to protein engineering and design of protein-protein interactions. PMID:18611374

Nuclear half-lives for {alpha}-radioactivity of elements with 100 {<=} Z {<=} 130

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chowdhury, P. Roy; Samanta, C.; Physics Department, Gottwald Science Center, University of Richmond, Richmond, VA 23173

2008-11-15

Theoretical estimates for the half-lives of about 1700 isotopes of heavy elements with 100 {<=} Z {<=} 130 are tabulated using theoretical Q-values. The quantum mechanical tunneling probabilities are calculated within a WKB framework using microscopic nuclear potentials. The microscopic nucleus-nucleus potentials are obtained by folding the densities of interacting nuclei with a density-dependent M3Y effective nucleon-nucleon interaction. The {alpha}-decay half-lives calculated in this formalism using the experimental Q-values were found to be in good agreement over a wide range of experimental data spanning about 20 orders of magnitude. The theoretical Q-values used for the present calculations are extracted frommore » three different mass estimates viz. Myers-Swiatecki, Muntian-Hofmann-Patyk-Sobiczewski, and Koura-Tachibana-Uno-Yamada.« less

Wang-Landau density of states based study of the folding-unfolding transition in the mini-protein Trp-cage (TC5b)

NASA Astrophysics Data System (ADS)

Singh, Priya; Sarkar, Subir K.; Bandyopadhyay, Pradipta

2014-07-01

We present the results of a high-statistics equilibrium study of the folding/unfolding transition for the 20-residue mini-protein Trp-cage (TC5b) in water. The ECEPP/3 force field is used and the interaction with water is treated by a solvent-accessible surface area method. A Wang-Landau type simulation is used to calculate the density of states and the conditional probabilities for the various values of the radius of gyration and the number of native contacts at fixed values of energy—along with a systematic check on their convergence. All thermodynamic quantities of interest are calculated from this information. The folding-unfolding transition corresponds to a peak in the temperature dependence of the computed specific heat. This is corroborated further by the structural signatures of folding in the distributions for radius of gyration and the number of native contacts as a function of temperature. The potentials of mean force are also calculated for these variables, both separately and jointly. A local free energy minimum, in addition to the global minimum, is found in a temperature range substantially below the folding temperature. The free energy at this second minimum is approximately 5 kBT higher than the value at the global minimum.

Mimicking the action of folding chaperones by Hamiltonian replica-exchange molecular dynamics simulations: application in the refinement of de novo models.

PubMed

Fan, Hao; Periole, Xavier; Mark, Alan E

2012-07-01

The efficiency of using a variant of Hamiltonian replica-exchange molecular dynamics (Chaperone H-replica-exchange molecular dynamics [CH-REMD]) for the refinement of protein structural models generated de novo is investigated. In CH-REMD, the interaction between the protein and its environment, specifically, the electrostatic interaction between the protein and the solvating water, is varied leading to cycles of partial unfolding and refolding mimicking some aspects of folding chaperones. In 10 of the 15 cases examined, the CH-REMD approach sampled structures in which the root-mean-square deviation (RMSD) of secondary structure elements (SSE-RMSD) with respect to the experimental structure was more than 1.0 Å lower than the initial de novo model. In 14 of the 15 cases, the improvement was more than 0.5 Å. The ability of three different statistical potentials to identify near-native conformations was also examined. Little correlation between the SSE-RMSD of the sampled structures with respect to the experimental structure and any of the scoring functions tested was found. The most effective scoring function tested was the DFIRE potential. Using the DFIRE potential, the SSE-RMSD of the best scoring structures was on average 0.3 Å lower than the initial model. Overall the work demonstrates that targeted enhanced-sampling techniques such as CH-REMD can lead to the systematic refinement of protein structural models generated de novo but that improved potentials for the identification of near-native structures are still needed. Copyright © 2012 Wiley Periodicals, Inc.

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

PubMed Central

Warepam, Marina; Sharma, Gurumayum Suraj; Dar, Tanveer Ali; Khan, Md. Khurshid Alam; Singh, Laishram Rajendrakumar

2014-01-01

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K m and k cat), thermodynamic stability (T m and ΔH m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes. PMID:25313668

Exploring the Sequence-based Prediction of Folding Initiation Sites in Proteins.

PubMed

Raimondi, Daniele; Orlando, Gabriele; Pancsa, Rita; Khan, Taushif; Vranken, Wim F

2017-08-18

Protein folding is a complex process that can lead to disease when it fails. Especially poorly understood are the very early stages of protein folding, which are likely defined by intrinsic local interactions between amino acids close to each other in the protein sequence. We here present EFoldMine, a method that predicts, from the primary amino acid sequence of a protein, which amino acids are likely involved in early folding events. The method is based on early folding data from hydrogen deuterium exchange (HDX) data from NMR pulsed labelling experiments, and uses backbone and sidechain dynamics as well as secondary structure propensities as features. The EFoldMine predictions give insights into the folding process, as illustrated by a qualitative comparison with independent experimental observations. Furthermore, on a quantitative proteome scale, the predicted early folding residues tend to become the residues that interact the most in the folded structure, and they are often residues that display evolutionary covariation. The connection of the EFoldMine predictions with both folding pathway data and the folded protein structure suggests that the initial statistical behavior of the protein chain with respect to local structure formation has a lasting effect on its subsequent states.

Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A-H2B Heterodimer

PubMed Central

Stump, Matthew R.; Gloss, Lisa M.

2010-01-01

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I2. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ~0.4 M urea (concentrations inaccessible from the 4 M urea unfolded state), a roll-over in the rates was observed; this suggests that a component of the I2 ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I2 ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I2 and the transition state between I2 and N2. This limited mutational study indicated that residues in the α2 helices of H2A and H2B, as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I2 burst phase species. Interestingly, at least eight of the nine targeted residues stabilize I2 by interactions that are non-native to some extent. Given that destabilizing I2 and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structure present in the I2 ensemble enables efficient folding of H2A-H2B. PMID:20600120

Self-emulsifying drug delivery systems: Design of a novel vaginal delivery system for curcumin.

PubMed

Köllner, S; Nardin, I; Markt, R; Griesser, J; Prüfert, F; Bernkop-Schnürch, A

2017-06-01

The aim of this study was to develop a vaginal self-emulsifying delivery system for curcumin being capable of spreading, of permeating the mucus gel layer and of protecting the drug being incorporated in oily nanodroplets towards mucus interactions and immobilization. The emulsifying properties of curcumin loaded SEDDS containing 30% Cremophor RH40, 20% Capmul PG-8, 30% Captex 300, 10% DMSO and 10% tetraglycol (SEDD formulation A) as well as 25% PEG 200, 35% Cremophor RH40, 20% Captex 355, 10% Caprylic acid and 10% Tween 80 (SEDD formulation B) after diluting 1+2 with artificial vaginal fluid were characterized regarding droplet size and zeta potential. Collagen swelling test was used to examine the irritation potential of SEDDS. Additionally to mucus binding studies, permeation studies in the mucus were performed. Furthermore, spreading potential of the novel developed formulations was compared with a commercial available o/w cream (non-ionic hydrophilic cream) on vaginal mucosa. SEDDS displayed a mean droplet size between 38 and 141nm and a zeta potential of -0.3 to -1.6mV. The collagen swelling test indicated no significant irritation potential of both formulations over 24h. An immediate interaction of unformulated curcumin with the mucus was determined, whereas both SEDDS facilitated drug permeation through the mucus layer. Formulation B showed a 2.2-fold improved transport ratio of curcumin compared to SEDD formulation A. In comparison to the vaginal cream, SEDD formulation A and B were able to spread over the vaginal mucosa and cover the tissue to a 17.8- and 14.8-fold higher extent, respectively. According to these results, SEDDS seems to be a promising tool for vaginal application. Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrogen bonding and interparticle forces in platelet alpha-Al2O3 dispersions: yield stress and zeta potential.

PubMed

Khoo, Kay-Sen; Teh, E-Jen; Leong, Yee-Kwong; Ong, Ban Choon

2009-04-09

Adsorbed phosphate on smooth platelet alpha-Al2O3 particles at saturation surface coverage gives rise to strong interparticle attractive forces in dispersion. The maximum yield stress at the point of zero charge was increased by 2-fold. This was attributed to a high density of intermolecular hydrogen bonding between the adsorbed phosphate layers of the interacting particles. Adsorbed citrate at saturation surface coverage, however, reduced the maximum yield stress by 50%. It adsorbed to form a very effective steric barrier as intramolecular hydrogen bonding between -OH and the free terminal carboxylic group prevented strong interactions with other adsorbed citrate molecules residing on the second interacting particle. This steric barrier kept the interacting platelet particles further apart, thereby weakening the van der Waals attraction. The platelet alpha-Al2O3 dispersions were flocculated at all pH level. These dispersions displayed a maximum yield stress at the point of zero zeta potential at the pH approximately 8.0. They also obeyed the yield stress-DLVO force model as characterized by a linear decrease in the yield stress with the square of the zeta potential.

R-citalopram functionally antagonises escitalopram in vivo and in vitro: evidence for kinetic interaction at the serotonin transporter

PubMed Central

Stórustovu, Signe í; Sánchez, Connie; Pörzgen, Peter; Brennum, Lise T; Larsen, Anna Kirstine; Pulis, Monica; Ebert, Bjarke

2004-01-01

Clinical observations with the selective serotonin reuptake inhibitor (SSRI), S-citalopram, indicate that S-citalopram is more efficacious and produces earlier symptom relief than RS-citalopram. Since R-citalopram is at least 20-fold weaker than S-citalopram as inhibitor of the 5-HT transporter (SERT) in preclinical studies, the clinical data suggest an unexpected antagonistic interaction between the two enantiomers. We therefore characterised the interaction of R- and S-citalopram with the SERT in in vivo and in vitro assays. In both behavioural (potentiation of 5-hydroxytryptophan (5-HTP)-induced behaviour) and electrophysiological studies (inhibition of 5-HT-elicited ion currents in Xenopus oocytes expressing the human SERT (hSERT) R-citalopram inhibited the effects of S-citalopram in a dose-dependent manner. With S-citalopram : R-citalopram ratios of 1 : 2 and 1 : 4, 5-HTP potentiation was significantly smaller than with S-citalopram alone. emsp;R-citalopram did not antagonise the effects of another SSRI (fluoxetine) in either behavioural or electrophysiological studies. In oocytes, inhibition of hSERT-mediated currents by R-citalopram was almost completely reversible and characterised by fast on- and off-sets of action. In contrast, the off-set for S-citalopram was 35-fold slower than for R-citalopram. Kinetic analysis of the oocyte experiments suggests that S-citalopram binding to SERT induces a long-lasting, inhibited state of the transporter and that coapplication of R-citalopram partially relieves SERT of this persistent inhibition. We propose that the kinetic interaction of R- and S-citalopram with SERT is a critical factor contributing to the antagonistic effects of R-citalopram on S-citalopram in vitro and in vivo. PMID:15037515

Studies of protein-protein and protein-water interactions by small angle x-ray scattering, terahertz spectroscopy, ASMOS, and computer simulation

NASA Astrophysics Data System (ADS)

Kim, Seung Joong

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 microm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

NASA Astrophysics Data System (ADS)

Iacovache, Ioan; de Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît

2016-07-01

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

The structure of a conserved Piezo channel domain reveals a novel beta sandwich fold

PubMed Central

Kamajaya, Aron; Kaiser, Jens; Lee, Jonas; Reid, Michelle; Rees, Douglas C.

2014-01-01

Summary Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a novel beta sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in Dehydrated Hereditary Stomatocytosis (DHS) patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels. PMID:25242456

The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold.

PubMed

Kamajaya, Aron; Kaiser, Jens T; Lee, Jonas; Reid, Michelle; Rees, Douglas C

2014-10-07

Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels. Copyright © 2014 Elsevier Ltd. All rights reserved.

An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors

PubMed Central

Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G.; Tawfik, Dan S.

2016-01-01

Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose’s ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint—geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif. PMID:26938925

The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PubMed

Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

2016-06-01

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

A strong enrichment of potentially toxic elements (PTEs) in Nord-Trøndelag (central Norway) forest soil.

PubMed

Reimann, C; Fabian, K; Schilling, J; Roberts, D; Englmaier, P

2015-12-01

Analysis of soil C and O horizon samples in a recent regional geochemical survey of Nord-Trøndelag, central Norway (752 sample sites covering 25,000 km2), identified a strong enrichment of several potentially toxic elements (PTEs) in the O horizon. Of 53 elements analysed in both materials, Cd concentrations are, on average, 17 times higher in the O horizon than in the C horizon and other PTEs such as Ag (11-fold), Hg (10-fold), Sb (8-fold), Pb (4-fold) and Sn (2-fold) are all strongly enriched relative to the C horizon. Geochemical maps of the survey area do not reflect an impact from local or distant anthropogenic contamination sources in the data for O horizon soil samples. The higher concentrations of PTEs in the O horizon are the result of the interaction of the underlying geology, the vegetation zone and type, and climatic effects. Based on the general accordance with existing data from earlier surveys in other parts of northern Europe, the presence of a location-independent, superordinate natural trend towards enrichment of these elements in the O horizon relative to the C horizon soil is indicated. The results imply that the O and C horizons of soils are different geochemical entities and that their respective compositions are controlled by different processes. Local mineral soil analyses (or published data for the chemical composition of the average continental crust) cannot be used to provide a geochemical background for surface soil. At the regional scale used here surface soil chemistry is still dominated by natural sources and processes. Copyright © 2015 Elsevier B.V. All rights reserved.

Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation.

PubMed

Goldberg, M E; Expert-Bezançon, N; Vuillard, L; Rabilloud, T

1996-01-01

Attempts to renature proteins often yield aggregates rather than native protein. To minimize aggregation, low protein concentrations and/or solubilizing agents are used. Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial beta-D-galactosidase. The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured. The five sulphobetaines improved the yield of native lysozyme up to 12-fold. Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold. Non-detergent sulphobetaines strongly affect the balance between aggregation and folding. Their effect depends on their structure and on their interactions with folding intermediates. These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines.

Folding and stability of helical bundle proteins from coarse-grained models.

PubMed

Kapoor, Abhijeet; Travesset, Alex

2013-07-01

We develop a coarse-grained model where solvent is considered implicitly, electrostatics are included as short-range interactions, and side-chains are coarse-grained to a single bead. The model depends on three main parameters: hydrophobic, electrostatic, and side-chain hydrogen bond strength. The parameters are determined by considering three level of approximations and characterizing the folding for three selected proteins (training set). Nine additional proteins (containing up to 126 residues) as well as mutated versions (test set) are folded with the given parameters. In all folding simulations, the initial state is a random coil configuration. Besides the native state, some proteins fold into an additional state differing in the topology (structure of the helical bundle). We discuss the stability of the native states, and compare the dynamics of our model to all atom molecular dynamics simulations as well as some general properties on the interactions governing folding dynamics. Copyright © 2013 Wiley Periodicals, Inc.

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein, musashi1, as revealed by comparison of their structures, modes of interaction, surface electrostatic potentials, and backbone dynamics.

PubMed

Miyanoiri, Youhei; Kobayashi, Hisanori; Imai, Takao; Watanabe, Michinao; Nagata, Takashi; Uesugi, Seiichi; Okano, Hideyuki; Katahira, Masato

2003-10-17

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.

Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.

PubMed

Kukic, Predrag; Kannan, Arvind; Dijkstra, Maurits J J; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele

2015-10-01

It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a 'tube model' approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the 'CamTube' force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost.

Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations

PubMed Central

Dijkstra, Maurits J. J.; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele

2015-01-01

It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a ‘tube model’ approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the ‘CamTube’ force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost. PMID:26505754

Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.

PubMed

Therien, J P Daniel; Baenziger, John E

2017-03-27

Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.

Mimicking coarse-grained simulations without coarse-graining: enhanced sampling by damping short-range interactions.

PubMed

Wei, Dongshan; Wang, Feng

2010-08-28

The damped-short-range-interaction (DSRI) method is proposed to mimic coarse-grained simulations by propagating an atomistic scale system on a smoothed potential energy surface. The DSRI method has the benefit of enhanced sampling provided by a typical coarse-grained simulation without the need to perform coarse-graining. Our method was used to simulate liquid water, alanine dipeptide folding, and the self-assembly of dimyristoylphosphatidylcholine lipid. In each case, our method appreciably accelerated the dynamics without significantly changing the free energy surface. Additional insights from DSRI simulations and the promise of coupling our DSRI method with Hamiltonian replica-exchange molecular dynamics are discussed.

Mimicking coarse-grained simulations without coarse-graining: Enhanced sampling by damping short-range interactions

NASA Astrophysics Data System (ADS)

Wei, Dongshan; Wang, Feng

2010-08-01

The damped-short-range-interaction (DSRI) method is proposed to mimic coarse-grained simulations by propagating an atomistic scale system on a smoothed potential energy surface. The DSRI method has the benefit of enhanced sampling provided by a typical coarse-grained simulation without the need to perform coarse-graining. Our method was used to simulate liquid water, alanine dipeptide folding, and the self-assembly of dimyristoylphosphatidylcholine lipid. In each case, our method appreciably accelerated the dynamics without significantly changing the free energy surface. Additional insights from DSRI simulations and the promise of coupling our DSRI method with Hamiltonian replica-exchange molecular dynamics are discussed.

Specific interactions between amyloid-β peptides in an amyloid-β hexamer with three-fold symmetry: Ab initio fragment molecular orbital calculations in water

NASA Astrophysics Data System (ADS)

Ishimura, Hiromi; Tomioka, Shogo; Kadoya, Ryushi; Shimamura, Kanako; Okamoto, Akisumi; Shulga, Sergiy; Kurita, Noriyuki

2017-03-01

The accumulation of amyloid-beta (Aβ) aggregates in brain contributes to the onset of Alzheimer's disease (AD). Recent structural analysis for the tissue obtained from AD patients revealed that Aβ aggregates have a single structure with three-fold symmetry. To explain why this structure possesses significant stability, we here investigated the specific interactions between Aβ peptides in the aggregate, using ab initio fragment molecular orbital calculations. The results indicate that the interactions between the Aβ peptides of the stacked Aβ pair are stronger than those between the Aβ peptides of the trimer with three-fold symmetry and that the charged amino-acids are important.

Prediction of atrial fibrillation recurrence after cardioversion-interaction analysis of cardiac autonomic regulation.

PubMed

Seeck, A; Rademacher, W; Fischer, C; Haueisen, J; Surber, R; Voss, A

2013-03-01

Today atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice accounting for approximately one third of hospitalizations and accompanied with a 5 fold increased risk for ischemic stroke and a 1.5 fold increased mortality risk. The role of the cardiac regulation system in AF recurrence after electrical cardioversion (CV) is still unclear. The aim of this study was to investigate the autonomic regulation by analyzing the interaction between heart rate and blood pressure using novel methods of nonlinear interaction dynamics, namely joint symbolic dynamics (JSD) and segmented Poincaré plot analysis (SPPA). For the first time, we applied SPPA to analyze the interaction between two time series. Introducing a parameter set of two indices, one derived from JSD and one from SPPA, the linear discriminant function analysis revealed an overall accuracy of 89% (sensitivity 91.7%, specificity 86.7%) for the classification between patients with stable sinus rhythm (group SR, n = 15) and with AF recurrence (group REZ, n = 12). This study proves that the assessment of the autonomic regulation by analyzing the coupling of heart rate and systolic blood pressure provides a potential tool for the prediction of AF recurrence after CV and could aid in the adjustment of therapeutic options for patients with AF. Copyright © 2012 IPEM. Published by Elsevier Ltd. All rights reserved.

Investigation of the pharmacokinetic interactions of deferasirox, a once-daily oral iron chelator, with midazolam, rifampin, and repaglinide in healthy volunteers.

PubMed

Skerjanec, Andrej; Wang, Jixian; Maren, Kelly; Rojkjaer, Lisa

2010-02-01

Deferasirox, a newly developed iron chelator, was coadministered orally with either a known inducer of drug metabolism or with cosubstrates for cytochrome P450 (CYP) to characterize the potential for drug-drug interactions. In the induction assessment, single-dose deferasirox pharmacokinetics were obtained in the presence and absence of a repeated-dose regimen of rifampin. In the CYP3A interaction evaluation, midazolam and its active hydroxylated metabolite were assessed after single doses of midazolam in the presence and absence of steady-state concentrations of deferasirox. To test for interaction at the level of CPY2C8, single-dose repaglinide pharmacokinetics/pharmacodynamics were determined with and without repeated-dose administration of deferasirox. After rifampin, a significant reduction (44%) in plasma exposure (AUC) to deferasirox was observed. Upon coadministration of midazolam, there was a modest reduction of up to 22% in midazolam exposure (AUC, C(max)), suggesting a modest induction of CYP3A4/5 by deferasirox. Def erasirox caused increases in repaglinide plasma C(max) and AUC of 1.5-fold to over 2-fold, respectively, with little change in blood glucose measures. Specific patient prescribing recommendations were established when coadministering deferasirox with midazolam, repaglinide, and rifampin. These recommendations may also apply to other substrates of CYP3A4/5 and CYP2C8 or potent inducers of glucuronidation.

Interaction intensity and pollinator-mediated selection.

PubMed

Trunschke, Judith; Sletvold, Nina; Ågren, Jon

2017-05-01

In animal-pollinated plants, the opportunity for selection and the strength of pollinator-mediated selection are expected to increase with the degree of pollen limitation. However, whether differences in pollen limitation can explain variation in pollinator-mediated and net selection among animal-pollinated species is poorly understood. In the present study, we quantified pollen limitation, variance in relative fitness and pollinator-mediated selection on five traits important for pollinator attraction (flowering start, plant height, flower number, flower size) and pollination efficiency (spur length) in natural populations of 12 orchid species. Pollinator-mediated selection was quantified by subtracting estimates of selection gradients for plants receiving supplemental hand-pollination from estimates obtained for open-pollinated control plants. Mean pollen limitation ranged from zero to 0.96. Opportunity for selection, pollinator-mediated selection and net selection were all positively related to pollen limitation, whereas nonpollinator-mediated selection was not. Opportunity for selection varied five-fold, strength of pollinator-mediated selection varied three-fold and net selection varied 1.5-fold among species. Supplemental hand-pollination reduced both opportunity for selection and selection on floral traits. The results show that the intensity of biotic interactions is an important determinant of the selection regime, and indicate that the potential for pollinator-mediated selection and divergence in floral traits is particularly high in species that are strongly pollen-limited. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Tannic Acid as a Potential Modulator of Norfloxacin Resistance in Staphylococcus Aureus Overexpressing norA.

PubMed

Diniz-Silva, Helena Taina; Cirino, Isis Caroline da Silva; Falcão-Silva, Vivyanne Dos Santos; Magnani, Marciane; de Souza, Evandro Leite; Siqueira-Júnior, José P

2016-01-01

Tannins have shown inhibitory effects against pathogenic bacteria, and these properties make tannins potential modifying agents in bacterial resistance. The minimum inhibitory concentration (MIC) of tannic acid (TA), gallic acid (GA) and norfloxacin (Nor) against Staphylococcus aureus SA-1119 (NorA-effluxing strain) was determined using broth microdilution tests. To assess the modulation of antibiotic resistance, the MIC of Nor was determined in growth media with or without TA or GA at a subinhibitory concentration (1/4 MIC). The checkerboard method was performed to obtain the fractional inhibitory concentration index (FICI) for the combined application of TA and Nor. TA displayed a weak inhibitory effect (MIC 512 μg/ml) against S. aureus SA-1119, while no inhibitory effect was displayed by GA (MIC >512 μg/ml). However, when TA was tested at a subinhibitory concentration in combination with Nor, the MIC of Nor against S. aureus SA-1119 decreased from 128 to 4 μg/ml (32-fold); this effect was not observed for GA. In the checkerboard assay, the MIC of TA and Nor decreased from 512 to 128 μg/ml (4-fold) and from 128 to 8 μg/ml (16-fold), respectively. The combination of TA and Nor presented an FICI as low as 0.31, which indicates a synergistic interaction. TA is a potential agent for increasing the clinical efficacy of Nor to control resistant S. aureus. © 2016 S. Karger AG, Basel.

ΛcN interaction from lattice QCD and its application to Λc hypernuclei

NASA Astrophysics Data System (ADS)

Miyamoto, Takaya; Aoki, Sinya; Doi, Takumi; Gongyo, Shinya; Hatsuda, Tetsuo; Ikeda, Yoichi; Inoue, Takashi; Iritani, Takumi; Ishii, Noriyoshi; Kawai, Daisuke; Murano, Keiko; Nemura, Hidekatsu; Sasaki, Kenji

2018-03-01

The interaction between Λc and a nucleon (N) is investigated by employing the HAL QCD method in the (2 + 1)-flavor lattice QCD on a (2.9fm) 3 volume at mπ ≃ 410 , 570 , 700 MeV. We study the central potential in S10 channel as well as central and tensor potentials in S31-3D1 channel, and find that the tensor potential for Λc N is negligibly weak and central potentials in both S10 and S31-3D1 channels are almost identical with each other except at short distances. Phase shifts and scattering lengths calculated with these potentials show that the interaction of Λc N system is attractive and has a similar strength in S10 and S31 channels at low energies (i.e. the kinetic energy less than about 40 MeV). While the attractions are not strong enough to form two-body bound states, our results lead to a possibility to form Λc hypernuclei for sufficiently large atomic numbers (A). To demonstrate this, we derive a single-folding potential for Λc hypernuclei from the Λc-nucleon potential obtained in lattice QCD, and find that Λc hypernuclei can exist for A ≥ 12 with the binding energies of a few MeV. We also estimate the Coulomb effect for the Λc hypernuclei.

Substantiating In Vivo Magnetic Brain Tumor Targeting of Cationic Iron Oxide Nanocarriers via Adsorptive Surface Masking

PubMed Central

Chertok, Beata; David, Allan E.; Moffat, Bradford A.; Yang, Victor C.

2009-01-01

Cationic magnetic nanoparticles are attractive as potential vehicles for tumor drug delivery due to their favorable interactions with both the tumor milieu and the therapeutic cargo. However, systemic delivery of these nanoparticles to the tumor site is compromised by their rapid plasma clearance. We developed a simple method for in vivo protection of cationic nanocarriers, using non-covalent surface masking with a conjugate of low molecular weight heparin and polyethylene glycol. Surface masking resulted in an 11-fold increase in plasma AUC and a 2-fold increase in the magnetic capture of systemically injected nanoparticles in orthotopic rodent brain tumors. Overall, the described methodology could expand the prospective applications for cationic magnetic nanoparticles in magnetically-mediated gene/drug delivery. PMID:19782394

Statistical analysis of native contact formation in the folding of designed model proteins

NASA Astrophysics Data System (ADS)

Tiana, Guido; Broglia, Ricardo A.

2001-02-01

The time evolution of the formation probability of native bonds has been studied for designed sequences which fold fast into the native conformation. From this analysis a clear hierarchy of bonds emerge: (a) local, fast forming highly stable native bonds built by some of the most strongly interacting amino acids of the protein; (b) nonlocal bonds formed late in the folding process, in coincidence with the folding nucleus, and involving essentially the same strongly interacting amino acids already participating in the fast bonds; (c) the rest of the native bonds whose behavior is subordinated, to a large extent, to that of the strong local and nonlocal native contacts.

Fluid-Structure Interactions with Flexible and Rigid Bodies

NASA Astrophysics Data System (ADS)

Daily, David Jesse

Fluid structure interactions occur to some extent in nearly every type of fluid flow. Understanding how structures interact with fluids and visa-versa is of vital importance in many engineering applications. The purpose of this research is to explore how fluids interact with flexible and rigid structures. A computational model was used to model the fluid structure interactions of vibrating synthetic vocal folds. The model simulated the coupling of the fluid and solid domains using a fluid-structure interface boundary condition. The fluid domain used a slightly compressible flow solver to allow for the possibility of acoustic coupling with the subglottal geometry and vibration of the vocal fold model. As the subglottis lengthened, the frequency of vibration decreased until a new acoustic mode could form in the subglottis. Synthetic aperture particle image velocimetry (SAPIV) is a three-dimensional particle tracking technique. SAPIV was used to image the jet of air that emerges from vibrating human vocal folds (glottal jet) during phonation. The three-dimensional reconstruction of the glottal jet found faint evidence of flow characteristics seen in previous research, such as axis-switching, but did not have sufficient resolution to detect small features. SAPIV was further applied to reconstruct the smaller flow characteristics of the glottal jet of vibrating synthetic vocal folds. Two- and four-layer synthetic vocal fold models were used to determine how the glottal jet from the synthetic models compared to the glottal jet from excised human vocal folds. The two- and four-layer models clearly exhibited axis-switching which has been seen in other 3D analyses of the glottal jet. Cavitation in a quiescent fluid can break a rigid structure such as a glass bottle. A new cavitation number was derived to include acceleration and pressure head at cavitation onset. A cavitation stick was used to validate the cavitation number by filling it with different depths and hitting the stick to cause fluid cavitation. Acceleration was measured using an accelerometer and cavitation bubbles were detected using a high-speed camera. Cavitation in an accelerating fluid occurred at a cavitation number of 1. Keywords: Fluid structure interaction, vocal folds, acoustics, SAPIV, cavitation, slightly compressible

Grapefruit juice and its constituents augment colchicine intestinal absorption: potential hazardous interaction and the role of p-glycoprotein.

PubMed

Dahan, Arik; Amidon, Gordon L

2009-04-01

To investigate the potential interaction between grapefruit juice (GFJ) and the oral microtubule polymerization inhibitor colchicine, a P-gp and CYP3A4 substrate. Colchicine intestinal epithelial transport was investigated across Caco-2 cell monolayers in both AP-BL and BL-AP directions, in the absence/presence of known P-gp inhibitors (verapamil and quinidine). The concentration-dependent effects of GFJ and its major constituents (6'-7'-dihydroxybergamottin, naringin and naringenin) on colchicine Caco-2 mucosal secretion were examined. The effect of GFJ on colchicine intestinal-permeability was then investigated in-situ in the rat perfusion model, in both jejunum and ileum. Colchicine exhibited 20-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion, which was reduced by verapamil/quinidine. Colchicine AP-BL permeability was increased and BL-AP was decreased by GFJ in a concentration-dependent manner (IC(50) values of 0.75% and 0.46% respectively), suggesting inhibition of efflux transport, rather than metabolizing enzyme. Similar effects obtained following pre-experiment incubation with GFJ, even though the juice was not present throughout the transepithelial study. 6'-7'-Dihydroxybergamottin, naringin and naringenin displayed concentration-dependent inhibition on colchicine BL-AP secretion (IC(50) values of 90, 592 and 11.6 microM respectively). Ten percent GFJ doubled colchicine rat in-situ ileal permeability, and increased 1.5-fold jejunal permeability. The data suggest that GFJ may augment colchicine oral bioavailability. Due to colchicine narrow therapeutic-index and severely toxic side-effects, awareness of this interaction is prudent.

Quantum mechanical electronic structure calculation reveals orientation dependence of hydrogen bond energy in proteins.

PubMed

Mondal, Abhisek; Datta, Saumen

2017-06-01

Hydrogen bond plays a unique role in governing macromolecular interactions with exquisite specificity. These interactions govern the fundamental biological processes like protein folding, enzymatic catalysis, molecular recognition. Despite extensive research work, till date there is no proper report available about the hydrogen bond's energy surface with respect to its geometric parameters, directly derived from proteins. Herein, we have deciphered the potential energy landscape of hydrogen bond directly from the macromolecular coordinates obtained from Protein Data Bank using quantum mechanical electronic structure calculations. The findings unravel the hydrogen bonding energies of proteins in parametric space. These data can be used to understand the energies of such directional interactions involved in biological molecules. Quantitative characterization has also been performed using Shannon entropic calculations for atoms participating in hydrogen bond. Collectively, our results constitute an improved way of understanding hydrogen bond energies in case of proteins and complement the knowledge-based potential. Proteins 2017; 85:1046-1055. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

PubMed Central

Ioannou, Petros; Andrianaki, Aggeliki; Akoumianaki, Tonia; Kyrmizi, Irene; Albert, Nathaniel; Perlin, David; Samonis, George

2015-01-01

The modest in vitro activity of echinocandins against Aspergillus implies that host-related factors augment the action of these antifungal agents in vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against various Aspergillus species under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P = 0. 0005). Importantly, the enhanced activity of caspofungin against Aspergillus spp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinating Aspergillus hyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin with Aspergillus hyphae (P < 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery to Aspergillus hyphae. PMID:26643329

Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1986-07-31

Neuromedin N (NN) is a novel neurotensin (NT)-like hexapeptide recently isolated from porcine spinal cord. NN competitively inhibited the binding of monoiodinated [Trp11]NT to rat brain synaptic membranes with a 19-fold lower potency than NT. In the presence of 1 mM 1,10-phenanthroline or 10 microM bestatin, the potency of NN relative to NT was increased about 5-fold. NN was readily degraded by rat brain synaptic membranes, and NN-(2-6) was the major degradation product. NN-(2-6) did not bind to NT receptors at concentrations up to 1 microM whether or not peptidase inhibitors were present in the binding assay. The rate of degradation by synaptic membranes was nearly 2.5 times higher for NN than for NT. NN degradation by membranes was totally prevented by 1,10-phenanthroline and markedly inhibited by bestatin. The presence of NN in the central nervous system, its high potency to interact with brain NT receptors and its rapid inactivation by brain synaptic peptidases make it a potential neurotransmitter candidate acting at the NT receptor.

7Li(15N, 14C)8Be reaction at 81 MeV and 14C + 8Be interaction versus that of 13C + 8Be

NASA Astrophysics Data System (ADS)

Rudchik, A. T.; Rudchik, A. A.; Muravynets, L. M.; Kemper, K. W.; Rusek, K.; Koshchy, E. I.; Piasecki, E.; Trzcinska, A.; Pirnak, Val. M.; Ponkratenko, O. A.; Strojek, I.; Stolarz, A.; Plujko, V. A.; Sakuta, S. B.; Siudak, R.; Ilyin, A. P.; Stepanenko, Yu. M.; Shyrma, Yu. O.; Uleshchenko, V. V.

2018-03-01

Angular distributions of the 7Li(15N, 14C)8Be reaction were measured at the energy Elab(15N) = 81 MeV. Data for transfer to the ground and first two excited states in 8Be were acquired as well as to the 14C ground and excited states. The reaction data were analyzed within the coupled-reaction-channels (CRC) method. The required 15N + 7Li entrance channel potential was taken from the 15N + 7Li elastic scattering. The 14C + 8Be potential was found by fitting Woods-Saxon form potentials to those generated by double folded real and imaginary potentials in the region of interaction. These generated potentials were then used in the CRC calculations. Proton transfer dominants this reaction, including to the excited states of 8Be. The reaction dependence on the exit channel potential was examined by using the 13C + 8Be potential previously deduced from the 9Be(12C, 13C)8Be reaction and 14C + 8Be from the 13C(9Be, 8Be)14C reaction.

Peppytides: Interactive Models of Polypeptide Chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zuckermann, Ron; Chakraborty, Promita; Derisi, Joe

2014-01-21

Peppytides are scaled, 3D-printed models of polypeptide chains that can be folded into accurate protein structures. Designed and created by Berkeley Lab Researcher, Promita Chakraborty, and Berkeley Lab Senior Scientist, Dr. Ron Zuckermann, Peppytides are accurate physical models of polypeptide chains that anyone can interact with and fold intro various protein structures - proving to be a great educational tool, resulting in a deeper understanding of these fascinating structures and how they function. Build your own Peppytide model and learn about how nature's machines fold into their intricate architectures!

Consequences of localized frustration for the folding mechanism of the IM7 protein

PubMed Central

Sutto, Ludovico; Lätzer, Joachim; Hegler, Joseph A.; Ferreiro, Diego U.; Wolynes, Peter G.

2007-01-01

In the laboratory, IM7 has been found to have an unusual folding mechanism in which an “on-pathway” intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble. PMID:18077415

Peppytides: Interactive Models of Polypeptide Chains

ScienceCinema

Zuckermann, Ron; Chakraborty, Promita; Derisi, Joe

2018-06-08

Peppytides are scaled, 3D-printed models of polypeptide chains that can be folded into accurate protein structures. Designed and created by Berkeley Lab Researcher, Promita Chakraborty, and Berkeley Lab Senior Scientist, Dr. Ron Zuckermann, Peppytides are accurate physical models of polypeptide chains that anyone can interact with and fold intro various protein structures - proving to be a great educational tool, resulting in a deeper understanding of these fascinating structures and how they function. Build your own Peppytide model and learn about how nature's machines fold into their intricate architectures!

Comparing the energy landscapes for native folding and aggregation of PrP

PubMed Central

Dee, Derek R.; Woodside, Michael T.

2016-01-01

ABSTRACT Protein sequences are evolved to encode generally one folded structure, out of a nearly infinite array of possible folds. Underlying this code is a funneled free energy landscape that guides folding to the native conformation. Protein misfolding and aggregation are also a manifestation of free-energy landscapes. The detailed mechanisms of these processes are poorly understood, but often involve rare, transient species and a variety of different pathways. The inherent complexity of misfolding has hampered efforts to measure aggregation pathways and the underlying energy landscape, especially using traditional methods where ensemble averaging obscures important rare and transient events. We recently studied the misfolding and aggregation of prion protein by examining 2 monomers tethered in close proximity as a dimer, showing how the steps leading to the formation of a stable aggregated state can be resolved in the single-molecule limit and the underlying energy landscape thereby reconstructed. This approach allows a more quantitative comparison of native folding versus misfolding, including fundamental differences in the dynamics for misfolding. By identifying key steps and interactions leading to misfolding, it should help to identify potential drug targets. Here we describe the importance of characterizing free-energy landscapes for aggregation and the challenges involved in doing so, and we discuss how single-molecule studies can help test proposed structural models for PrP aggregates. PMID:27191683

Proteomic analysis of a decellularized human vocal fold mucosa scaffold using 2D electrophoresis and high-resolution mass spectrometry.

PubMed

Welham, Nathan V; Chang, Zhen; Smith, Lloyd M; Frey, Brian L

2013-01-01

Natural biologic scaffolds for tissue engineering are commonly generated by decellularization of tissues and organs. Despite some preclinical and clinical success, in vivo scaffold remodeling and functional outcomes remain variable, presumably due to the influence of unidentified bioactive molecules on the scaffold-host interaction. Here, we used 2D electrophoresis and high-resolution mass spectrometry-based proteomic analyses to evaluate decellularization effectiveness and identify potentially bioactive protein remnants in a human vocal fold mucosa model. We noted proteome, phosphoproteome and O-glycoproteome depletion post-decellularization, and identified >200 unique protein species within the decellularized scaffold. Gene ontology-based enrichment analysis revealed a dominant set of functionally-related ontology terms associated with extracellular matrix assembly, organization, morphology and patterning, consistent with preservation of a tissue-specific niche for later cell seeding and infiltration. We further identified a subset of ontology terms associated with bioactive (some of which are antigenic) cellular proteins, despite histological and immunohistochemical data indicating complete decellularization. These findings demonstrate the value of mass spectrometry-based proteomics in identifying agents potentially responsible for variation in host response to engineered tissues derived from decellularized scaffolds. This work has implications for the manufacturing of biologic scaffolds from any tissue or organ, as well as for prediction and monitoring of the scaffold-host interaction in vivo. Copyright © 2012 Elsevier Ltd. All rights reserved.

Theoretical Insights into the Biophysics of Protein Bi-stability and Evolutionary Switches

PubMed Central

Krobath, Heinrich; Chan, Hue Sun

2016-01-01

Deciphering the effects of nonsynonymous mutations on protein structure is central to many areas of biomedical research and is of fundamental importance to the study of molecular evolution. Much of the investigation of protein evolution has focused on mutations that leave a protein’s folded structure essentially unchanged. However, to evolve novel folds of proteins, mutations that lead to large conformational modifications have to be involved. Unraveling the basic biophysics of such mutations is a challenge to theory, especially when only one or two amino acid substitutions cause a large-scale conformational switch. Among the few such mutational switches identified experimentally, the one between the GA all-α and GB α+β folds is extensively characterized; but all-atom simulations using fully transferrable potentials have not been able to account for this striking switching behavior. Here we introduce an explicit-chain model that combines structure-based native biases for multiple alternative structures with a general physical atomic force field, and apply this construct to twelve mutants spanning the sequence variation between GA and GB. In agreement with experiment, we observe conformational switching from GA to GB upon a single L45Y substitution in the GA98 mutant. In line with the latent evolutionary potential concept, our model shows a gradual sequence-dependent change in fold preference in the mutants before this switch. Our analysis also indicates that a sharp GA/GB switch may arise from the orientation dependence of aromatic π-interactions. These findings provide physical insights toward rationalizing, predicting and designing evolutionary conformational switches. PMID:27253392

Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

NASA Technical Reports Server (NTRS)

Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

2015-01-01

Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modeling.

PubMed Central

Peelman, F.; Vinaimont, N.; Verhee, A.; Vanloo, B.; Verschelde, J. L.; Labeur, C.; Seguret-Mace, S.; Duverger, N.; Hutchinson, G.; Vandekerckhove, J.; Tavernier, J.; Rosseneu, M.

1998-01-01

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates. PMID:9541390

Potential artifacts associated with historical preparation of joint compound samples and reported airborne asbestos concentrations.

PubMed

Brorby, G P; Sheehan, P J; Berman, D W; Bogen, K T; Holm, S E

2011-05-01

Airborne samples collected in the 1970s for drywall workers using asbestos-containing joint compounds were likely prepared and analyzed according to National Institute of Occupational Safety and Health Method P&CAM 239, the historical precursor to current Method 7400. Experimentation with a re-created, chrysotile-containing, carbonate-based joint compound suggested that analysis following sample preparation by the historical vs. current method produces different fiber counts, likely because of an interaction between the different clearing and mounting chemicals used and the carbonate-based joint compound matrix. Differences were also observed during analysis using Method 7402, depending on whether acetic acid/dimethylformamide or acetone was used during preparation to collapse the filter. Specifically, air samples of sanded chrysotile-containing joint compound prepared by the historical method yielded fiber counts significantly greater (average of 1.7-fold, 95% confidence interval: 1.5- to 2.0-fold) than those obtained by the current method. In addition, air samples prepared by Method 7402 using acetic acid/dimethylformamide yielded fiber counts that were greater (2.8-fold, 95% confidence interval: 2.5- to 3.2-fold) than those prepared by this method using acetone. These results indicated (1) there is an interaction between Method P&CAM 239 preparation chemicals and the carbonate-based joint compound matrix that reveals fibers that were previously bound in the matrix, and (2) the same appeared to be true for Method 7402 preparation chemicals acetic acid/dimethylformamide. This difference in fiber counts is the opposite of what has been reported historically for samples of relatively pure chrysotile dusts prepared using the same chemicals. This preparation artifact should be considered when interpreting historical air samples for drywall workers prepared by Method P&CAM 239. Copyright © 2011 JOEH, LLC

The affinity of a major Ca2+ binding site on GRP78 is differentially enhanced by ADP and ATP.

PubMed

Lamb, Heather K; Mee, Christopher; Xu, Weiming; Liu, Lizhi; Blond, Sylvie; Cooper, Alan; Charles, Ian G; Hawkins, Alastair R

2006-03-31

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered approximately 11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered approximately 40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.

Interactions of the hepatitis C virus protease inhibitor faldaprevir with cytochrome P450 enzymes: in vitro and in vivo correlation.

PubMed

Sabo, John P; Kort, Jens; Ballow, Charles; Kashuba, Angela D M; Haschke, Manuel; Battegay, Manuel; Girlich, Birgit; Ting, Naitee; Lang, Benjamin; Zhang, Wei; Cooper, Curtis; O'Brien, Drané; Seibert, Eleanore; Chan, Tom S; Tweedie, Donald; Li, Yongmei

2015-04-01

The potential inhibition of the major human cytochrome P450 (CYP) enzymes by faldaprevir was evaluated both in vitro and in clinical studies (healthy volunteers and hepatitis C virus [HCV] genotype 1-infected patients). In vitro studies indicated that faldaprevir inhibited CYP2B6, CYP2C9, and CYP3A, and was a weak-to-moderate inactivator of CYP3A4. Faldaprevir 240 mg twice daily in healthy volunteers demonstrated moderate inhibition of hepatic and intestinal CYP3A (oral midazolam: 2.96-fold increase in AUC(0-24 h)), weak inhibition of hepatic CYP3A (intravenous midazolam: 1.56-fold increase in AUC(0-24 h)), weak inhibition of CYP2C9 ([S]-warfarin: 1.29-fold increase in AUC(0-120 h)), and had no relevant effects on CYP1A2, CYP2B6, or CYP2D6. Faldaprevir 120 mg once daily in HCV-infected patients demonstrated weak inhibition of hepatic and intestinal CYP3A (oral midazolam: 1.52-fold increase in AUC(0-∞)), and had no relevant effects on CYP2C9 or CYP1A2. In vitro drug-drug interaction predictions based on inhibitor concentration ([I])/inhibition constant (Ki) ratios tended to overestimate clinical effects and a net-effect model provided a more accurate approach. These studies suggest that faldaprevir shows a dose-dependent inhibition of CYP3A and CYP2C9, and does not induce CYP isoforms. © 2015, The American College of Clinical Pharmacology.

Confinement in nanopores can destabilize α-helix folding proteins and stabilize the β structures

NASA Astrophysics Data System (ADS)

Javidpour, Leili; Sahimi, Muhammad

2011-09-01

Protein folding in confined media has attracted wide attention over the past decade due to its importance in both in vivo and in vitro applications. Currently, it is generally believed that protein stability increases by decreasing the size of the confining medium, if its interaction with the confining walls is repulsive, and that the maximum folding temperature in confinement occurs for a pore size only slightly larger than the smallest dimension of the folded state of a protein. Protein stability in pore sizes, very close to the size of the folded state, has not however received the attention that it deserves. Using detailed, 0.3-ms-long molecular dynamics simulations, we show that proteins with an α-helix native state can have an optimal folding temperature in pore sizes that do not affect the folded-state structure. In contradiction to the current theoretical explanations, we find that the maximum folding temperature occurs in larger pores for smaller α-helices. In highly confined pores the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state. In addition, in small nanopores the protein states that contain the β structures are entropically stabilized, in contrast to the bulk. As a consequence, folding rates decrease notably and the free energy surface becomes rougher. The results shed light on many recent experimental observations that cannot be explained by the current theories, and demonstrate the importance of entropic effects on proteins' misfolded states in highly confined environments. They also support the concept of passive effect of chaperonin GroEL on protein folding by preventing it from aggregation in crowded environment of biological cells, and provide deeper clues to the α → β conformational transition, believed to contribute to Alzheimer's and Parkinson's diseases. The strategy of protein and enzyme stabilization in confined media may also have to be revisited in the case of tight confinement. For in silico studies of protein folding in confined media, use of non-Go potentials may be more appropriate.

Orientation-dependent potential of mean force for protein folding

NASA Astrophysics Data System (ADS)

Mukherjee, Arnab; Bhimalapuram, Prabhakar; Bagchi, Biman

2005-07-01

We present a solvent-implicit minimalistic model potential among the amino acid residues of proteins, obtained by using the known native structures [deposited in the Protein Data Bank (PDB)]. In this model, the amino acid side chains are represented by a single ellipsoidal site, defined by the group of atoms about the center of mass of the side chain. These ellipsoidal sites interact with other sites through an orientation-dependent interaction potential which we construct in the following fashion. First, the site-site potential of mean force (PMF) between heavy atoms is calculated [following F. Melo and E. Feytsman, J. Mol. Biol. 267, 207 (1997)] from statistics of their distance separation obtained from crystal structures. These site-site potentials are then used to calculate the distance and the orientation-dependent potential between side chains of all the amino acid residues (AAR). The distance and orientation dependencies show several interesting results. For example, we find that the PMF between two hydrophobic AARs, such as phenylalanine, is strongly attractive at short distances (after the obvious repulsive region at very short separation) and is characterized by a deep minimum, for specific orientations. For the interaction between two hydrophilic AARs, such a deep minimum is absent and in addition, the potential interestingly reveals the combined effect of polar (charge) and hydrophobic interactions among some of these AARs. The effectiveness of our potential has been tested by calculating the Z-scores for a large set of proteins. The calculated Z-scores show high negative values for most of them, signifying the success of the potential to identify the native structure from among a large number of its decoy states.

Adiabatic and coupled channels calculations for near barrier fusion of 16O +238U using realistic nucleon-nucleon interaction

NASA Astrophysics Data System (ADS)

Ismail, M.; Seif, W. M.; Botros, M. M.

2016-04-01

We investigate the fusion cross-section and the fusion barrier distribution of 16O +238U at near- and sub-barrier energies. We use an interaction potential generated by the semi-microscopic double folding model-based on density dependent (DD) form of the realistic Michigan-three-Yukawa (M3Y) Reid nucleon-nucleon (NN) interaction. We studied the role of both the static and dynamic deformations of the target nucleus on the fusion process. Rotational and vibrational degrees of freedom of 238U-nucleus are considered. We found that the deformation and the octupole vibrations in 238U enhance its sub-barrier fusion cross-section. The signature of the the octupole vibrational modes of 238U appears clearly in its fusion barrier distribution profile.

Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix

PubMed Central

Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K.; Karimi, Tahereh

2015-01-01

An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73–92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005–0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the total concentration. PMID:25051457

Exploration of the relationship between topology and designability of conformations

NASA Astrophysics Data System (ADS)

Leelananda, Sumudu P.; Towfic, Fadi; Jernigan, Robert L.; Kloczkowski, Andrzej

2011-06-01

Protein structures are evolutionarily more conserved than sequences, and sequences with very low sequence identity frequently share the same fold. This leads to the concept of protein designability. Some folds are more designable and lots of sequences can assume that fold. Elucidating the relationship between protein sequence and the three-dimensional (3D) structure that the sequence folds into is an important problem in computational structural biology. Lattice models have been utilized in numerous studies to model protein folds and predict the designability of certain folds. In this study, all possible compact conformations within a set of two-dimensional and 3D lattice spaces are explored. Complementary interaction graphs are then generated for each conformation and are described using a set of graph features. The full HP sequence space for each lattice model is generated and contact energies are calculated by threading each sequence onto all the possible conformations. Unique conformation giving minimum energy is identified for each sequence and the number of sequences folding to each conformation (designability) is obtained. Machine learning algorithms are used to predict the designability of each conformation. We find that the highly designable structures can be distinguished from other non-designable conformations based on certain graphical geometric features of the interactions. This finding confirms the fact that the topology of a conformation is an important determinant of the extent of its designability and suggests that the interactions themselves are important for determining the designability.

Frustration in Condensed Matter and Protein Folding

NASA Astrophysics Data System (ADS)

Li, Z.; Tanner, S.; Conroy, B.; Owens, F.; Tran, M. M.; Boekema, C.

2014-03-01

By means of computer modeling, we are studying frustration in condensed matter and protein folding, including the influence of temperature and Thomson-figure formation. Frustration is due to competing interactions in a disordered state. The key issue is how the particles interact to reach the lowest frustration. The relaxation for frustration is mostly a power function (randomly assigned pattern) or an exponential function (regular patterns like Thomson figures). For the atomic Thomson model, frustration is predicted to decrease with the formation of Thomson figures at zero kelvin. We attempt to apply our frustration modeling to protein folding and dynamics. We investigate the homogeneous protein frustration that would cause the speed of the protein folding to increase. Increase of protein frustration (where frustration and hydrophobicity interplay with protein folding) may lead to a protein mutation. Research is supported by WiSE@SJSU and AFC San Jose.

A budget of energy transfer in a sustained vocal folds vibration in glottis

NASA Astrophysics Data System (ADS)

Zhang, Lucy; Yang, Jubiao; Krane, Michael

2016-11-01

A set of force and energy balance equations using the control volume approach is derived based on the first principles of physics for a sustained vocal folds vibration in glottis. The control volume analysis is done for compressible airflow in a moving and deforming control volume in the vicinity of the vocal folds. The interaction between laryngeal airflow and vocal folds are successfully simulated using the modified Immersed Finite Element Method (mIFEM), a fully coupled approach to simulate fluid-structure interactions. Detailed mathematical terms are separated out for deeper physical understanding and utilization of mechanical energy is quantified with the derived equation. The results show that majority of energy input is consumed for driving laryngeal airflow, while a smaller portion is for compensating viscous losses in and sustaining the vibration of the vocal folds. We acknowledge the funding support of NIH 2R01DC005642-10A1.

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax*S⃞

PubMed Central

Liu, Ying; Matthews, Kathleen S.; Bondos, Sarah E.

2008-01-01

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding ∼2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further ∼40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions. PMID:18508761

Optimization of protease-inhibitor interactions by randomizing adventitious contacts

PubMed Central

Komiyama, Tomoko; VanderLugt, Bryan; Fugère, Martin; Day, Robert; Kaufman, Randal J.; Fuller, Robert S.

2003-01-01

Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c. Two kinds of contacts exist in such complexes: (i) reactive site loop-active site contacts and (ii) interactions outside of these that form the broader enzyme-inhibitor interface. We hypothesized that the second class of “adventitious” contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases. We began with a modified eglin c, Arg-42–Arg-45–eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family. We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin. Substitutions at one of these sites, Y49, were also screened against yeast Kex2 and human PC7. These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin). One variant, Asp-49–Arg-42–Arg-45–eglin, exhibited a Ki of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells. The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein–protein interactions. PMID:12832612

Equilibrium denaturation and preferential interactions of an RNA tetraloop with urea

DOE PAGES

Miner, Jacob Carlson; García, Angel Enrique

2017-02-09

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarilymore » show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Lastly, our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.« less

Equilibrium Denaturation and Preferential Interactions of an RNA Tetraloop with Urea.

PubMed

Miner, Jacob C; García, Angel E

2017-04-20

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarily show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.

Equilibrium denaturation and preferential interactions of an RNA tetraloop with urea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miner, Jacob Carlson; García, Angel Enrique

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarilymore » show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Lastly, our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.« less

Atomic interaction networks in the core of protein domains and their native folds.

PubMed

Soundararajan, Venkataramanan; Raman, Rahul; Raguram, S; Sasisekharan, V; Sasisekharan, Ram

2010-02-23

Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be "signature" of a domain's native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1-2 angstroms (mean 1.61A) C(alpha) RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the 'twilight' and 'midnight' zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence-identity-independent applications demonstrated in this work, we suggest that the PCAIN is a fundamental fold feature that could be a valuable addition to the arsenal of protein modeling and analysis tools.

Atomic Interaction Networks in the Core of Protein Domains and Their Native Folds

PubMed Central

Soundararajan, Venkataramanan; Raman, Rahul; Raguram, S.; Sasisekharan, V.; Sasisekharan, Ram

2010-01-01

Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be “signature” of a domain's native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1–2 angstroms (mean 1.61A) Cα RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the ‘twilight’ and ‘midnight’ zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence-identity-independent applications demonstrated in this work, we suggest that the PCAIN is a fundamental fold feature that could be a valuable addition to the arsenal of protein modeling and analysis tools. PMID:20186337

Folding dynamics of linear emulsion polymers into 3D architectures

NASA Astrophysics Data System (ADS)

McMullen, Angus; Bargteil, Dylan; Brujic, Jasna

Colloidal polymers have been limited to inflexible, solid colloids. Here we show that the fluidity of emulsion droplets allows for the self-assembly of flexible droplet chains, which can subsequently be folded into 3D structures via secondary interactions. We achieve this using DNA-guided interactions, to initially form the chain, and then program its folding pathways. When two emulsion droplets labeled with complementary DNA meet, the balance of hybridization energy and droplet deformation yields an equilibrium patch size. Therefore, the concentration of DNA on the surface determines the number of droplet-droplet bonds in the assembly. We find that 96 % of bound droplets successfully self-assemble into chains. Droplet binding is a stochastic process, following a Poisson distribution of lengths. Since the fluid droplets can rearrange, we compare the dynamics of emulsion chains to that of polymers. We also trigger secondary interactions along the chain, causing the formation of specific loops or compact clusters. This approach will allow us to fold our emulsion polymers into a wide array of soft structures, giving us a powerful biomimetic colloidal system to investigate protein folding on the mesoscopic scale. This work was supported by the NSF MRSEC Program (DMR-0820341).

In vitro-in vivo extrapolation of zolpidem as a perpetrator of metabolic interactions involving CYP3A.

PubMed

Polasek, Thomas M; Sadagopal, Janani S; Elliot, David J; Miners, John O

2010-03-01

To evaluate zolpidem as a mechanism-based inactivator of human CYP3A in vitro, and to assess its metabolic interaction potential with CYP3A drugs (in vitro-in vivo extrapolation; IV-IVE). A co- vs. pre-incubation strategy was used to quantify time-dependent inhibition of human liver microsomal (HLM) and recombinant CYP3A4 (rCYP3A4) by zolpidem. Experiments involving a 10-fold dilution step were employed to determine the kinetic constants of inactivation (K (I) and k (inact)) and to assess the in vitro mechanism-based inactivation (MBI) criteria. Inactivation data were entered into the Simcyp population-based ADME simulator to predict the increase in the area under the plasma concentration-time curve (AUC) for orally administered midazolam. Consistent with MBI, the inhibitory potency of zolpidem toward CYP3A was increased following pre-incubation. In HLMs, the concentration required for half maximal inactivation (K (I)) was 122 microM and the maximal rate of inactivation (k (inact)) was 0.094 min(-1). In comparison, K (I) and k (inact) values with rCYP3A4 were 50 microM and 0.229 min(-1), respectively. Zolpidem fulfilled all other in vitro MBI criteria, including irreversible inhibition. The mean oral AUC for midazolam in healthy volunteers was predicted to increase 1.1- to 1.7-fold due to the inhibition of metabolic clearance by zolpidem. Elderly subjects were more sensitive to the interaction, with mean increases in midazolam AUC of 1.2- and 2.2-fold for HLM IV-IVE and rCYP3A4 IV-IVE, respectively. Zolpidem is a relatively weak mechanism-based inactivator of human CYP3A in vitro. Zolpidem is unlikely to act as a significant perpetrator of metabolic interactions involving CYP3A.

Quantifying Functional Group Interactions that Determine Urea Effects on Nucleic Acid Helix Formation

PubMed Central

Guinn, Emily J.; Schwinefus, Jeffrey J.; Cha, Hyo Keun; McDevitt, Joseph L.; Merker, Wolf E.; Ritzer, Ryan; Muth, Gregory W.; Engelsgjerd, Samuel W.; Mangold, Kathryn E.; Thompson, Perry J.; Kerins, Michael J.; Record, Thomas

2013-01-01

Urea destabilizes helical and folded conformations of nucleic acids and proteins, as well as protein-nucleic acid complexes. To understand these effects, extend previous characterizations of interactions of urea with protein functional groups, and thereby develop urea as a probe of conformational changes in protein and nucleic acid processes, we obtain chemical potential derivatives (μ23 = dμ2/dm3) quantifying interactions of urea (component 3) with nucleic acid bases, base analogs, nucleosides and nucleotide monophosphates (component 2) using osmometry and hexanol-water distribution assays. Dissection of these μ23 yields interaction potentials quantifying interactions of urea with unit surface areas of nucleic acid functional groups (heterocyclic aromatic ring, ring methyl, carbonyl and phosphate O, amino N, sugar (C,O)); urea interacts favorably with all these groups, relative to interactions with water. Interactions of urea with heterocyclic aromatic rings and attached methyl groups (as on thymine) are particularly favorable, as previously observed for urea-homocyclic aromatic ring interactions. Urea m-values determined for double helix formation by DNA dodecamers near 25°C are in the range 0.72 to 0.85 kcal mol−1 m−1 and exhibit little systematic dependence on nucleobase composition (17–42% GC). Interpretation of these results using the urea interaction potentials indicates that extensive (60–90%) stacking of nucleobases in the separated strands in the transition region is required to explain the m-value. Results for RNA and DNA dodecamers obtained at higher temperatures, and literature data, are consistent with this conclusion. This demonstrates the utility of urea as a quantitative probe of changes in surface area (ΔASA) in nucleic acid processes. PMID:23510511

Models of S/π interactions in protein structures: Comparison of the H2S–benzene complex with PDB data

PubMed Central

Ringer, Ashley L.; Senenko, Anastasia; Sherrill, C. David

2007-01-01

S/π interactions are prevalent in biochemistry and play an important role in protein folding and stabilization. Geometries of cysteine/aromatic interactions found in crystal structures from the Brookhaven Protein Data Bank (PDB) are analyzed and compared with the equilibrium configurations predicted by high-level quantum mechanical results for the H2S–benzene complex. A correlation is observed between the energetically favorable configurations on the quantum mechanical potential energy surface of the H2S–benzene model and the cysteine/aromatic configurations most frequently found in crystal structures of the PDB. In contrast to some previous PDB analyses, configurations with the sulfur over the aromatic ring are found to be the most important. Our results suggest that accurate quantum computations on models of noncovalent interactions may be helpful in understanding the structures of proteins and other complex systems. PMID:17766371

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strickland, Madeleine; Stanley, Ann Marie; Wang, Guangshun

Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EIsugar and NPr:EINtr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EIsugar interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EINtr complex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domainmore » of EINtr (EINNtr), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.« less

Structural Determinants of Sleeping Beauty Transposase Activity

PubMed Central

Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

2016-01-01

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called “sectors”, which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold. PMID:27401040

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Quadruplexes in 'Dicty': crystal structure of a four-quartet G-quadruplex formed by G-rich motif found in the Dictyostelium discoideum genome.

PubMed

Guédin, Aurore; Lin, Linda Yingqi; Armane, Samir; Lacroix, Laurent; Mergny, Jean-Louis; Thore, Stéphane; Yatsunyk, Liliya A

2018-06-01

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249-1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson-Crick base pair and a A-T-A triad and displays high thermal stability (Tm > 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools.

Modulation of opioid analgesia by agmatine.

PubMed

Kolesnikov, Y; Jain, S; Pasternak, G W

1996-01-18

Administered alone, agmatine at doses of 0.1 or 10 mg/kg is without effect in the mouse tailflick assay. However, agmatine enhances morphine analgesia in a dose-dependent manner, shifting morphine's ED50 over 5-fold. A far greater effect is observed when morphine is given intrathecally (9-fold shift) than after intracerebroventricular administration (2-fold). In contrast to the potentiation of morphine analgesia, agmatine (10 mg/kg) has no effect on morphine's inhibition of gastrointestinal transit. delta-Opioid receptor-mediated analgesia also is potentiated by agmatine, but kappa1-receptor-mediated (U50,488H; trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetemide) and kappa3-opioid receptor-mediated (naloxone benzoylhydrazone) analgesia is not significantly enhanced by any dose of agmatine tested in this acute model. In chronic studies, agmatine at a low dose (0.1 mg/kg) which does not affect morphine analgesia acutely prevents tolerance following chronic morphine dosing for 10 days. A higher agmatine dose (10 mg/kg) has a similar effect. Agmatine also blocks tolerance to the delta-opioid receptor ligand [D-Pen2,D-Pen5]enkephalin given intrathecally, but not to the kappa3-opioid receptor agonist naloxone benzoylhydrazone. Despite its inactivity on kappa1-opioid analgesia in the acute model, agmatine prevents kappa1-opioid receptor-mediated tolerance. These studies demonstrate the dramatic interactions between agmatine and opioid analgesia and tolerance.

The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures

PubMed Central

Komives, Elizabeth A.; Wolynes, Peter G.

2008-01-01

Repeat-proteins are made up of near repetitions of 20– to 40–amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi–one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete “domain” (the stability and cooperativity of the repeating array) can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (ΔGwater) and the cooperativity of denaturation (m-value), which do not ordinarily apply for globular proteins. We show how the parameters for the “coarse-grained” description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR) repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are “poised” at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions. PMID:18483553

Blocking the Metastatic Phenotype in Prostate Cancer Through Direct Inhibition of the ETV/PEA3 Transcription Factors

DTIC Science & Technology

2016-11-01

the ortho phenolic aldehyde , a carboxylic acid positioned meta or para to the ring junction, and small hydrophobic groups along the rim of the rings...estimate potential toxicity risks, as outlined in the original proposal and SOW. The phenolic aldehyde present in all three of the first...in the absence of this interaction, however, with an approximately 8-fold shift in IC50. Consistent with this result, reduction of the aldehyde in

Molecular chaperones and protein folding as therapeutic targets in Parkinson's disease and other synucleinopathies.

PubMed

Ebrahimi-Fakhari, Darius; Saidi, Laiq-Jan; Wahlster, Lara

2013-12-05

Changes in protein metabolism are key to disease onset and progression in many neurodegenerative diseases. As a prime example, in Parkinson's disease, folding, post-translational modification and recycling of the synaptic protein α-synuclein are clearly altered, leading to a progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. Altered protein folding is one of the first steps of an increasingly understood cascade in which α-synuclein forms complex oligomers and finally distinct protein aggregates, termed Lewy bodies and Lewy neurites. In neurons, an elaborated network of chaperone and co-chaperone proteins is instrumental in mediating protein folding and re-folding. In addition to their direct influence on client proteins, chaperones interact with protein degradation pathways such as the ubiquitin-proteasome-system or autophagy in order to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins. Because of the vital role of proper protein folding for protein homeostasis, a growing number of studies have evaluated the contribution of chaperone proteins to neurodegeneration. We herein review our current understanding of the involvement of chaperones, co-chaperones and chaperone-mediated autophagy in synucleinopathies with a focus on the Hsp90 and Hsp70 chaperone system. We discuss genetic and pathological studies in Parkinson's disease as well as experimental studies in models of synucleinopathies that explore molecular chaperones and protein degradation pathways as a novel therapeutic target. To this end, we examine the capacity of chaperones to prevent or modulate neurodegeneration and summarize the current progress in models of Parkinson's disease and related neurodegenerative disorders.

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

PubMed

Dogan, Jakob; Jonasson, Josefin; Andersson, Eva; Jemth, Per

2015-08-04

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions.

PubMed

Ramirez-Sarmiento, Cesar A; Komives, Elizabeth A

2018-04-06

Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction. Copyright © 2018 Elsevier Inc. All rights reserved.

Insights on fluid-rock interaction evolution during deformation from fracture network geochemistry at reservoir-scale

NASA Astrophysics Data System (ADS)

Beaudoin, Nicolas; Koehn, Daniel; Lacombe, Olivier; Bellahsen, Nicolas; Emmanuel, Laurent

2015-04-01

Fluid migration and fluid-rock interactions during deformation is a challenging problematic to picture. Numerous interplays, as between porosity-permeability creation and clogging, or evolution of the mechanical properties of rock, are key features when it comes to monitor reservoir evolution, or to better understand seismic cycle n the shallow crust. These phenomenoms are especially important in foreland basins, where various fluids can invade strata and efficiently react with limestones, altering their physical properties. Stable isotopes (O, C, Sr) measurements and fluid inclusion microthermometry of faults cement and veins cement lead to efficient reconstruction of the origin, temperature and migration pathways for fluids (i.e. fluid system) that precipitated during joints opening or faults activation. Such a toolbox can be used on a diffuse fracture network that testifies the local and/or regional deformation history experienced by the rock at reservoir-scale. This contribution underlines the advantages and limits of geochemical studies of diffuse fracture network at reservoir-scale by presenting results of fluid system reconstruction during deformation in folded structures from various thrust-belts, tectonic context and deformation history. We compare reconstructions of fluid-rock interaction evolution during post-deposition, post-burial growth of basement-involved folds in the Sevier-Laramide American Rocky Mountains foreland, a reconstruction of fluid-rock interaction evolution during syn-depostion shallow detachment folding in the Southern Pyrenean foreland, and a preliminary reconstruction of fluid-rock interactions in a post-deposition, post-burial development of a detachment fold in the Appenines. Beyond regional specification for the nature of fluids, a common behavior appears during deformation as in every fold, curvature-related joints (related either to folding or to foreland flexure) connected vertically the pre-existing stratified fluid system. The lengthscale of the migration and the nature of invading fluids during these connections is different in every studied example, and can be related to the tectonic nature of the fold, along with the burial depth at the time of deformation. Thus, to decipher fluid-fracture relationships provides insights to better reconstruct the mechanisms of deformation at reservoir-scale.

RNA-Seq reveals the molecular mechanism of trapping and killing of root-knot nematodes by nematode-trapping fungi.

PubMed

Pandit, Ramesh; Patel, Reena; Patel, Namrata; Bhatt, Vaibhav; Joshi, Chaitanya; Singh, Pawan Kumar; Kunjadia, Anju

2017-04-01

Nematode-trapping fungi are well known for their inherent potential to trap and kill nematodes using specialized trapping devices. However, the molecular mechanisms underlying the trapping and subsequent processes are still unclear. Therefore, in this study, we examined differential genes expression in two nematode-trapping fungi after baiting with nematode extracts. In Arthrobotrys conoides, 809 transcripts associated with diverse functions such as signal transduction, morphogenesis, stress response and peroxisomal proteins, proteases, chitinases and genes involved in the host-pathogen interaction showed differential expression with fold change (>±1.5 fold) in the presence of nematode extract with FDR (p-value < 0.001). G-proteins and mitogen activated protein kinases are considered crucial for signal transduction mechanism. Results of qRT-PCR of 20 genes further validated the sequencing data. Further, variations in gene expression among Duddingtonia flagrans and A. conoides showed septicity of nematode-trapping fungi for its host. The findings illustrate the molecular mechanism of fungal parasitism in A. conoides which may be helpful in developing a potential biocontrol agent against parasitic nematodes.

Age related changes in fractional elimination pathways for drugs: assessing the impact of variable ontogeny on metabolic drug-drug interactions.

PubMed

Salem, Farzaneh; Johnson, Trevor N; Barter, Zoe E; Leeder, J Steven; Rostami-Hodjegan, Amin

2013-08-01

The magnitude of any metabolic drug-drug interactions (DDIs) depends on fractional importance of inhibited pathway which may not necessarily be the same in young children when compared to adults. The ontogeny pattern of cytochrome P450 (CYP) enzymes (CYPs 1A2, 2B6, 2C8, 2C9, 2C18/19, 2D6, 2E1, 3A4) and renal function were analyzed systematically. Bootstrap methodology was used to account for variability, and to define the age range over which statistical differences existed between each pair of specific pathways. A number of DDIs were simulated (Simcyp Pediatric v12) for virtual compounds to highlight effects of age on fractional elimination and consequent magnitude of DDI. For a theoretical drug metabolized 50% by each of CYP2D6 and CYP3A4 pathways at birth, co-administration of ketoconazole (3 mg/kg) resulted in a 1.65-fold difference between inhibited versus uninhibited AUC compared to 2.4-fold in 1 year olds and 3.2-fold in adults. Conversely, neonates could be more sensitive to DDI than adults in certain scenarios. Thus, extrapolation from adult data may not be applicable across all pediatric age groups. The use of pediatric physiologically based pharmacokinetic (p-PBPK) models may offer an interim solution to uncovering potential periods of vulnerability to DDI where there are no existing clinical data derived from children. © The Author(s) 2013.

Alteration of the intravenous and oral pharmacokinetics of valsartan via the concurrent use of gemfibrozil in rats.

PubMed

Yang, Seung Jun; Kim, Bong Jin; Mo, Lingxuan; Han, Hyo-Kyung

2016-07-01

The present study aimed to examine the potential pharmacokinetic drug interaction between valsartan and gemfibrozil. Compared with the control given valsartan (10 mg/kg) alone, the concurrent use of gemfibrozil (10 mg/kg) significantly (p < 0.05) increased the oral exposure of valsartan in rats. In the presence of gemfibrozil, the Cmax and AUC of oral valsartan increased by 1.7- and 2.5-fold, respectively. Consequently, the oral bioavailability of valsartan was significantly higher (p < 0.05) in the presence of gemfibrozil compared with that of the control group. Furthermore, the intravenous pharmacokinetics of valsartan (1 mg/kg) was also altered by pretreatment with oral gemfibrozil (10 mg/kg). The plasma clearance of valsartan was decreased by two-fold in the presence of gemfibrozil, while the plasma half-life was not altered. In contrast, both the oral and intravenous pharmacokinetics of gemfibrozil were not affected by the concurrent use of valsartan. The cellular uptake of valsartan and gemfibrozil was also investigated by using cells overexpressing OATP1B1 or OATP1B3. Gemfibrozil and gemfibrozil 1-O-β glucuronide inhibited the cellular uptake of valsartan with IC50 values (µm) of 39.3 and 20.4, respectively, in MDCK/OATP1B1, while they were less interactive with OATP1B3. The cellular uptake of gemfibrozil was not affected by co-incubation with valsartan in both cells. Taken together, the present study suggests the potential drug interaction between valsartan and gemfibrozil, at least in part, via the OATP1B1-mediated transport pathways during hepatic uptake. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Impact of higher alginate expression on deposition of Pseudomonas aeruginosa in radial stagnation point flow and reverse osmosis systems.

PubMed

Herzberg, Moshe; Rezene, Tesfalem Zere; Ziemba, Christopher; Gillor, Osnat; Mathee, Kalai

2009-10-01

Extracellular polymeric substances (EPS) have major impact on biofouling of reverse osmosis (RO) membranes. On one hand, EPS can reduce membrane permeability and on the other, EPS production by the primary colonizers may influence their deposition and attachment rate and subsequently affect the biofouling propensity of the membrane. The role of bacterial exopolysaccharides in bacterial deposition followed by the biofouling potential of an RO membrane was evaluated using an alginate overproducing (mucoid) Pseudomonas aeruginosa. The mucoid P. aeruginosa PAOmucA22 was compared with its isogenic nonmucoid prototypic parent PAO1 microscopically in a radial stagnation point flow (RSPF) system for their bacterial deposition characteristics. Then, biofouling potential of PAO1 and PAOmucA22 was determined in a crossflow rectangular plate-and-frame membrane cell, in which the strains were cultivated on a thin-film composite, polyamide, flat RO membrane coupon (LFC-1) under laminar flow conditions. In the RSPF system, the observed deposition rate of the mucoid strain was between 5- and 10-fold lower than of the wild type using either synthetic wastewater medium (with ionic strength of 14.7 mM and pH 7.4) or 15 mM KCl solution (pH of 6.2). The slower deposition rate of the mucoid strain is explained by 5- to 25-fold increased hydrophilicity of the mucoid strain as compared to the isogenic wild type, PAO1. Corroborating with these results, a significant delay in the onset of biofouling of the RO membrane was observed when the mucoid strain was used as the membrane colonizer, in which the observed time for the induced permeate flux decline was delayed (ca. 2-fold). In conclusion, the lower initial cell attachment of the mucoid strain decelerated biofouling of the RO membrane. Bacterial deposition and attachment is a critical step in biofilm formation and governed by intimate interactions between outer membrane proteins of the bacteria and the surface. Shielding these interactions by a hydrated and hydrophilic alginate capsule is shown to dramatically lessen the biofouling potential of the membrane colonizers.

M1 RNA is important for the in-cell solubility of its cognate C5 protein: Implications for RNA-mediated protein folding

PubMed Central

Son, Ahyun; Choi, Seong Il; Han, Gyoonhee; Seong, Baik L

2015-01-01

It is one of the fundamental questions in biology how proteins efficiently fold into their native conformations despite off-pathway events such as misfolding and aggregation in living cells. Although molecular chaperones have been known to assist the de novo folding of certain types of proteins, the role of a binding partner (or a ligand) in the folding and in-cell solubility of its interacting protein still remains poorly defined. RNase P is responsible for the maturation of tRNAs as adaptor molecules of amino acids in ribosomal protein synthesis. The RNase P from Escherichia coli, composed of M1 RNA and C5 protein, is a prototypical ribozyme in which the RNA subunit contains the catalytic activity. Using E. coli RNase P, we demonstrate that M1 RNA plays a pivotal role in the in-cell solubility of C5 protein both in vitro and in vivo. Mutations in either the C5 protein or M1 RNA that affect their interactions significantly abolished the folding of C5 protein. Moreover, we find that M1 RNA provides quality insurance of interacting C5 protein, either by promoting the degradation of C5 mutants in the presence of functional proteolytic machinery, or by abolishing their solubility if the machinery is non-functional. Our results describe a crucial role of M1 RNA in the folding, in-cell solubility, and, consequently, the proteostasis of the client C5 protein, giving new insight into the biological role of RNAs as chaperones and mediators that ensure the quality of interacting proteins. PMID:26517763

Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.

PubMed

Teilum, Kaare; Thormann, Thorsten; Caterer, Nigel R; Poulsen, Heidi I; Jensen, Peter H; Knudsen, Jens; Kragelund, Birthe B; Poulsen, Flemming M

2005-04-01

Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues. (c) 2005 Wiley-Liss, Inc.

Sequentially distant but structurally similar proteins exhibit fold specific patterns based on their biophysical properties.

PubMed

Rajendran, Senthilnathan; Jothi, Arunachalam

2018-05-16

The Three-dimensional structure of a protein depends on the interaction between their amino acid residues. These interactions are in turn influenced by various biophysical properties of the amino acids. There are several examples of proteins that share the same fold but are very dissimilar at the sequence level. For proteins to share a common fold some crucial interactions should be maintained despite insignificant sequence similarity. Since the interactions are because of the biophysical properties of the amino acids, we should be able to detect descriptive patterns for folds at such a property level. In this line, the main focus of our research is to analyze such proteins and to characterize them in terms of their biophysical properties. Protein structures with sequence similarity lesser than 40% were selected for ten different subfolds from three different mainfolds (according to CATH classification) and were used for this analysis. We used the normalized values of the 49 physio-chemical, energetic and conformational properties of amino acids. We characterize the folds based on the average biophysical property values. We also observed a fold specific correlational behavior of biophysical properties despite a very low sequence similarity in our data. We further trained three different binary classification models (Naive Bayes-NB, Support Vector Machines-SVM and Bayesian Generalized Linear Model-BGLM) which could discriminate mainfold based on the biophysical properties. We also show that among the three generated models, the BGLM classifier model was able to discriminate protein sequences coming under all beta category with 81.43% accuracy and all alpha, alpha-beta proteins with 83.37% accuracy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural Insights into DD-Fold Assembly and Caspase-9 Activation by the Apaf-1 Apoptosome.

PubMed

Su, Tsung-Wei; Yang, Chao-Yu; Kao, Wen-Pin; Kuo, Bai-Jiun; Lin, Shan-Meng; Lin, Jung-Yaw; Lo, Yu-Chih; Lin, Su-Chang

2017-03-07

Death domain (DD)-fold assemblies play a crucial role in regulating the signaling to cell survival or death. Here we report the crystal structure of the caspase recruitment domain (CARD)-CARD disk of the human apoptosome. The structure surprisingly reveals that three 1:1 Apaf-1:procaspase-9 CARD protomers form a novel helical DD-fold assembly on the heptameric wheel-like platform of the apoptosome. The small-angle X-ray scattering and multi-angle light scattering data also support that three protomers could form an oligomeric complex similar to the crystal structure. Interestingly, the quasi-equivalent environment of CARDs could generate different quaternary CARD assemblies. We also found that the type II interaction is conserved in all DD-fold complexes, whereas the type I interaction is found only in the helical DD-fold assemblies. This study provides crucial insights into the caspase activation mechanism, which is tightly controlled by a sophisticated and highly evolved CARD assembly on the apoptosome, and also enables better understanding of the intricate DD-fold assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

PubMed Central

Betts, S. D.; King, J.

1998-01-01

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly. PMID:9684883

Vocal fold contact patterns based on normal modes of vibration.

PubMed

Smith, Simeon L; Titze, Ingo R

2018-05-17

The fluid-structure interaction and energy transfer from respiratory airflow to self-sustained vocal fold oscillation continues to be a topic of interest in vocal fold research. Vocal fold vibration is driven by pressures on the vocal fold surface, which are determined by the shape of the glottis and the contact between vocal folds. Characterization of three-dimensional glottal shapes and contact patterns can lead to increased understanding of normal and abnormal physiology of the voice, as well as to development of improved vocal fold models, but a large inventory of shapes has not been directly studied previously. This study aimed to take an initial step toward characterizing vocal fold contact patterns systematically. Vocal fold motion and contact was modeled based on normal mode vibration, as it has been shown that vocal fold vibration can be almost entirely described by only the few lowest order vibrational modes. Symmetric and asymmetric combinations of the four lowest normal modes of vibration were superimposed on left and right vocal fold medial surfaces, for each of three prephonatory glottal configurations, according to a surface wave approach. Contact patterns were generated from the interaction of modal shapes at 16 normalized phases during the vibratory cycle. Eight major contact patterns were identified and characterized by the shape of the flow channel, with the following descriptors assigned: convergent, divergent, convergent-divergent, uniform, split, merged, island, and multichannel. Each of the contact patterns and its variation are described, and future work and applications are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Multi-parameter machine learning approach to the neuroanatomical basis of developmental dyslexia.

PubMed

Płoński, Piotr; Gradkowski, Wojciech; Altarelli, Irene; Monzalvo, Karla; van Ermingen-Marbach, Muna; Grande, Marion; Heim, Stefan; Marchewka, Artur; Bogorodzki, Piotr; Ramus, Franck; Jednoróg, Katarzyna

2017-02-01

Despite decades of research, the anatomical abnormalities associated with developmental dyslexia are still not fully described. Studies have focused on between-group comparisons in which different neuroanatomical measures were generally explored in isolation, disregarding potential interactions between regions and measures. Here, for the first time a multivariate classification approach was used to investigate grey matter disruptions in children with dyslexia in a large (N = 236) multisite sample. A variety of cortical morphological features, including volumetric (volume, thickness and area) and geometric (folding index and mean curvature) measures were taken into account and generalizability of classification was assessed with both 10-fold and leave-one-out cross validation (LOOCV) techniques. Classification into control vs. dyslexic subjects achieved above chance accuracy (AUC = 0.66 and ACC = 0.65 in the case of 10-fold CV, and AUC = 0.65 and ACC = 0.64 using LOOCV) after principled feature selection. Features that discriminated between dyslexic and control children were exclusively situated in the left hemisphere including superior and middle temporal gyri, subparietal sulcus and prefrontal areas. They were related to geometric properties of the cortex, with generally higher mean curvature and a greater folding index characterizing the dyslexic group. Our results support the hypothesis that an atypical curvature pattern with extra folds in left hemispheric perisylvian regions characterizes dyslexia. Hum Brain Mapp 38:900-908, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Drug interactions and risk of acute bleeding leading to hospitalisation or death in patients with chronic atrial fibrillation treated with warfarin.

PubMed

Gasse, Christiane; Hollowell, Jennifer; Meier, Christoph R; Haefeli, Walter E

2005-09-01

Although drug interactions with warfarin are an important cause of excessive anticoagulation, their impact on the risk of serious bleeding is unknown. We therefore performed a cohort study and a nested case-control analysis to determine the risk of serious bleeding in 4152 patients (aged 40-84 years) with nonvalvular atrial fibrillation (AF) taking long-term warfarin (> 3 months). The study population was drawn from the UK General Practice Research Database. More than half (58%) of eligible patients used potentially interacting drugs during continuous warfarin treatment. Among 45 identified cases of incident idiopathic bleeds (resulting in hospitalisation within 30 days or death within 7 days) and 143 matched controls, more cases than controls took > or = 1 potentially interacting drug within the preceding 30 days (62.2% vs. 35.7%) and used > 4 drugs (polypharmacy) within the preceding 90 days (80.0% vs. 66.4%). Conditional logistic regression analysis yielded an odds ratio (OR) of 3.4 (95% confidence interval [CI]: 1.4-8.5) for the risk of serious bleeding in patients treated with warfarin and > or = 1 drugs potentially increasing the effect of warfarin vs. warfarin alone adjusted for polypharmacy, diabetes, hypertension, heart failure, and thyroid disease; the adjusted OR for the combined use of warfarin and aspirin vs. warfarin alone was 4.5 (95% CI: 1.1-18.1). We conclude that concurrent use of potentially interacting drugs with warfarin is associated with a 3 to 4.5-fold increased risk of serious bleeding in long-term warfarin users.

Three-wave resonant interactions: Multi-dark-dark-dark solitons, breathers, rogue waves, and their interactions and dynamics

NASA Astrophysics Data System (ADS)

Zhang, Guoqiang; Yan, Zhenya; Wen, Xiao-Yong

2018-03-01

We investigate three-wave resonant interactions through both the generalized Darboux transformation method and numerical simulations. Firstly, we derive a simple multi-dark-dark-dark-soliton formula through the generalized Darboux transformation. Secondly, we use the matrix analysis method to avoid the singularity of transformed potential functions and to find the general nonsingular breather solutions. Moreover, through a limit process, we deduce the general rogue wave solutions and give a classification by their dynamics including bright, dark, four-petals, and two-peaks rogue waves. Ever since the coexistence of dark soliton and rogue wave in non-zero background, their interactions naturally become a quite appealing topic. Based on the N-fold Darboux transformation, we can derive the explicit solutions to depict their interactions. Finally, by performing extensive numerical simulations we can predict whether these dark solitons and rogue waves are stable enough to propagate. These results can be available for several physical subjects such as fluid dynamics, nonlinear optics, solid state physics, and plasma physics.

The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation.

PubMed

Xiao, Yucheng; Blumenthal, Kenneth; Jackson, James O; Liang, Songping; Cummins, Theodore R

2010-12-01

The voltage-gated sodium channel Na(v)1.7 plays a crucial role in pain, and drugs that inhibit hNa(v)1.7 may have tremendous therapeutic potential. ProTx-II and huwentoxin-IV (HWTX-IV), cystine knot peptides from tarantula venoms, preferentially block hNa(v)1.7. Understanding the interactions of these toxins with sodium channels could aid the development of novel pain therapeutics. Whereas both ProTx-II and HWTX-IV have been proposed to preferentially block hNa(v)1.7 activation by trapping the domain II voltage-sensor in the resting configuration, we show that specific residues in the voltage-sensor paddle of domain II play substantially different roles in determining the affinities of these toxins to hNa(v)1.7. The mutation E818C increases ProTx-II's and HWTX-IV's IC(50) for block of hNa(v)1.7 currents by 4- and 400-fold, respectively. In contrast, the mutation F813G decreases ProTx-II affinity by 9-fold but has no effect on HWTX-IV affinity. It is noteworthy that we also show that ProTx-II, but not HWTX-IV, preferentially interacts with hNa(v)1.7 to impede fast inactivation by trapping the domain IV voltage-sensor in the resting configuration. Mutations E1589Q and T1590K in domain IV each decreased ProTx-II's IC(50) for impairment of fast inactivation by ~6-fold. In contrast mutations D1586A and F1592A in domain-IV increased ProTx-II's IC(50) for impairment of fast inactivation by ~4-fold. Our results show that whereas ProTx-II and HWTX-IV binding determinants on domain-II may overlap, domain II plays a much more crucial role for HWTX-IV, and contrary to what has been proposed to be a guiding principle of sodium channel pharmacology, molecules do not have to exclusively target the domain IV voltage-sensor to influence sodium channel inactivation.

Aryl-Aryl Interactions in Designed Peptide Folds: Spectroscopic Characteristics and Placement Issues for Optimal Structure Stabilization

PubMed Central

Anderson, Jordan M.; Kier, Brandon; Jurban, Brice; Byrne, Aimee; Shu, Irene; Eidenschink, Lisa A.; Shcherbakov, Alexander A.; Hudson, Mike; Fesinmeyer, R. M.; Andersen, Niels H.

2017-01-01

We have extended our studies of Trp/Trp to other Aryl/Aryl through-space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T-shape orientation. Designated as an edge-to-face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W≫Y≥F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225–232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y and F/Y clusters, is a negative feature at 197–200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp-cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross-strand aryl/aryl clusters for the design of stable β-sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts. PMID:26850220

In vitro-in vivo extrapolation of CYP2D6 inactivation by paroxetine: prediction of nonstationary pharmacokinetics and drug interaction magnitude.

PubMed

Venkatakrishnan, Karthik; Obach, R Scott

2005-06-01

Attempts at predicting drug-drug interactions perpetrated by paroxetine from in vitro data have utilized reversible enzyme inhibition models and have been unsuccessful to date, grossly underpredicting interaction magnitude. Recent data have provided evidence for mechanism-based inactivation of CYP2D6 by paroxetine. We have predicted the pharmacokinetic consequences of CYP2D6 inactivation by paroxetine from in vitro inactivation kinetics (kinact 0.17 min(-1), unbound KI 0.315 microM), in vivo inhibitor concentrations, and an estimated CYP2D6 degradation half-life of 51 h, using a mathematical model of mechanism-based inhibition. The model-predicted accumulation ratio of paroxetine was 5 times that expected from single-dose kinetics and in excellent agreement with the observed 5- to 6-fold greater accumulation. Magnitudes of interactions produced by paroxetine (20-30 mg/day) with desipramine, risperidone, perphenazine, atomoxetine, (S)-metoprolol, and (R)-metoprolol were predicted, considering the contribution of CYP2D6 to their oral clearance. Predicted fold-increases in victim drug AUC were 5-, 6-, 5-, 6-, 4-, and 6-fold, respectively, and are in reasonable agreement with observed values of 5-, 6-, >7-, 7-, 5-, and 8-fold, respectively. Failure to consider microsomal binding in vitro adversely affected predictive accuracy. Simulation of the sensitivities of these predictions to model inputs suggests a 2-fold underprediction of interaction magnitude when a CYP2D6 degradation half-life of 14 h (reported for rat CYP3A) is used. In summary, the scaling model for mechanism-based inactivation successfully predicted the pharmacokinetic consequences of CYP2D6 inactivation by paroxetine from in vitro data.

Self-Complementarity within Proteins: Bridging the Gap between Binding and Folding

PubMed Central

Basu, Sankar; Bhattacharyya, Dhananjay; Banerjee, Rahul

2012-01-01

Complementarity, in terms of both shape and electrostatic potential, has been quantitatively estimated at protein-protein interfaces and used extensively to predict the specific geometry of association between interacting proteins. In this work, we attempted to place both binding and folding on a common conceptual platform based on complementarity. To that end, we estimated (for the first time to our knowledge) electrostatic complementarity (Em) for residues buried within proteins. Em measures the correlation of surface electrostatic potential at protein interiors. The results show fairly uniform and significant values for all amino acids. Interestingly, hydrophobic side chains also attain appreciable complementarity primarily due to the trajectory of the main chain. Previous work from our laboratory characterized the surface (or shape) complementarity (Sm) of interior residues, and both of these measures have now been combined to derive two scoring functions to identify the native fold amid a set of decoys. These scoring functions are somewhat similar to functions that discriminate among multiple solutions in a protein-protein docking exercise. The performances of both of these functions on state-of-the-art databases were comparable if not better than most currently available scoring functions. Thus, analogously to interfacial residues of protein chains associated (docked) with specific geometry, amino acids found in the native interior have to satisfy fairly stringent constraints in terms of both Sm and Em. The functions were also found to be useful for correctly identifying the same fold for two sequences with low sequence identity. Finally, inspired by the Ramachandran plot, we developed a plot of Sm versus Em (referred to as the complementarity plot) that identifies residues with suboptimal packing and electrostatics which appear to be correlated to coordinate errors. PMID:22713576

Self-complementarity within proteins: bridging the gap between binding and folding.

PubMed

Basu, Sankar; Bhattacharyya, Dhananjay; Banerjee, Rahul

2012-06-06

Complementarity, in terms of both shape and electrostatic potential, has been quantitatively estimated at protein-protein interfaces and used extensively to predict the specific geometry of association between interacting proteins. In this work, we attempted to place both binding and folding on a common conceptual platform based on complementarity. To that end, we estimated (for the first time to our knowledge) electrostatic complementarity (Em) for residues buried within proteins. Em measures the correlation of surface electrostatic potential at protein interiors. The results show fairly uniform and significant values for all amino acids. Interestingly, hydrophobic side chains also attain appreciable complementarity primarily due to the trajectory of the main chain. Previous work from our laboratory characterized the surface (or shape) complementarity (Sm) of interior residues, and both of these measures have now been combined to derive two scoring functions to identify the native fold amid a set of decoys. These scoring functions are somewhat similar to functions that discriminate among multiple solutions in a protein-protein docking exercise. The performances of both of these functions on state-of-the-art databases were comparable if not better than most currently available scoring functions. Thus, analogously to interfacial residues of protein chains associated (docked) with specific geometry, amino acids found in the native interior have to satisfy fairly stringent constraints in terms of both Sm and Em. The functions were also found to be useful for correctly identifying the same fold for two sequences with low sequence identity. Finally, inspired by the Ramachandran plot, we developed a plot of Sm versus Em (referred to as the complementarity plot) that identifies residues with suboptimal packing and electrostatics which appear to be correlated to coordinate errors. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Minimal model for the secondary structures and conformational conversions in proteins

NASA Astrophysics Data System (ADS)

Imamura, Hideo

Better understanding of protein folding process can provide physical insights on the function of proteins and makes it possible to benefit from genetic information accumulated so far. Protein folding process normally takes place in less than seconds but even seconds are beyond reach of current computational power for simulations on a system of all-atom detail. Hence, to model and explore protein folding process it is crucial to construct a proper model that can adequately describe the physical process and mechanism for the relevant time scale. We discuss the reduced off-lattice model that can express _-helix and ?-hairpin conformations defined solely by a given sequence in order to investigate a protein folding mechanism of conformations such as a ?-hairpin and also to investigate conformational conversions in proteins. The first two chapters introduce and review essential concepts in protein folding modelling physical interaction in proteins, various simple models, and also review computational methods, in particular, the Metropolis Monte Carlo method, its dynamic interpretation and thermodynamic Monte Carlo algorithms. Chapter 3 describes the minimalist model that represents both _-helix and ?-sheet conformations using simple potentials. The native conformation can be specified by the sequence without particular conformational biases to a reference state. In Chapter 4, the model is used to investigate the folding mechanism of ?-hairpins exhaustively using the dynamic Monte Carlo and a thermodynamic Monte Carlo method an effcient combination of the multicanonical Monte Carlo and the weighted histogram analysis method. We show that the major folding pathways and folding rate depend on the location of a hydrophobic. The conformational conversions between _-helix and ?-sheet conformations are examined in Chapter 5 and 6. First, the conformational conversion due to mutation in a non-hydrophobic system and then the conformational conversion due to mutation with a hydrophobic pair at a different position at various temperatures are examined.

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature.

PubMed

Zako, Tamotsu; Banba, Shinya; Sahlan, Muhamad; Sakono, Masafumi; Terada, Naofumi; Yohda, Masafumi; Maeda, Mizuo

2010-01-01

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak. Copyright 2009 Elsevier Inc. All rights reserved.

Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

NASA Astrophysics Data System (ADS)

Avitabile, Concetta; D'Andrea, Luca Domenico; Romanelli, Alessandra

2014-03-01

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae.

PubMed

Ioannou, Petros; Andrianaki, Aggeliki; Akoumianaki, Tonia; Kyrmizi, Irene; Albert, Nathaniel; Perlin, David; Samonis, George; Kontoyiannis, Dimitrios P; Chamilos, Georgios

2015-12-07

The modest in vitro activity of echinocandins against Aspergillus implies that host-related factors augment the action of these antifungal agents in vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against various Aspergillus species under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P = 0. 0005). Importantly, the enhanced activity of caspofungin against Aspergillus spp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinating Aspergillus hyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin with Aspergillus hyphae (P < 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery to Aspergillus hyphae. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Gelatin-based Hydrogel Degradation and Tissue Interaction in vivo: Insights from Multimodal Preclinical Imaging in Immunocompetent Nude Mice.

PubMed

Tondera, Christoph; Hauser, Sandra; Krüger-Genge, Anne; Jung, Friedrich; Neffe, Axel T; Lendlein, Andreas; Klopfleisch, Robert; Steinbach, Jörg; Neuber, Christin; Pietzsch, Jens

2016-01-01

Hydrogels based on gelatin have evolved as promising multifunctional biomaterials. Gelatin is crosslinked with lysine diisocyanate ethyl ester (LDI) and the molar ratio of gelatin and LDI in the starting material mixture determines elastic properties of the resulting hydrogel. In order to investigate the clinical potential of these biopolymers, hydrogels with different ratios of gelatin and diisocyanate (3-fold (G10_LNCO3) and 8-fold (G10_LNCO8) molar excess of isocyanate groups) were subcutaneously implanted in mice (uni- or bilateral implantation). Degradation and biomaterial-tissue-interaction were investigated in vivo (MRI, optical imaging, PET) and ex vivo (autoradiography, histology, serum analysis). Multimodal imaging revealed that the number of covalent net points correlates well with degradation time, which allows for targeted modification of hydrogels based on properties of the tissue to be replaced. Importantly, the degradation time was also dependent on the number of implants per animal. Despite local mechanisms of tissue remodeling no adverse tissue responses could be observed neither locally nor systemically. Finally, this preclinical investigation in immunocompetent mice clearly demonstrated a complete restoration of the original healthy tissue.

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

PubMed

Scherer, Paul C; Ding, Yan; Liu, Zhiqing; Xu, Jing; Mao, Haibin; Barrow, James C; Wei, Ning; Zheng, Ning; Snyder, Solomon H; Rao, Feng

2016-03-29

The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

Counterion effects in protein nanoparticle electrostatic binding: a theoretical study.

PubMed

Ghosh, Goutam

2015-04-01

Effects of counterions on the folding conformation of proteins, bound electrostatically on the surface of charge-ligand functionalized nanoparticles, have been investigated based on the protein folding energy calculation. The folding energy of a protein has been taken as a sum of the short range interaction energies, like, the van der Waals attraction and the hydrogen bond energies, and the long range coulomb interaction energy. On electrostatic binding, counterions associated with surface ligands of nanoparticles diffuse into bound proteins through the medium of dispersion. As a result, bound proteins partially unfold, as observed in circular dichroism experiments, which has been realized using the "charge-dipole" and the "charge-induced dipole" interactions of counterions with polar and non-polar residues, respectively. The effect of counterions solvation in the dispersing medium, e.g., water, which causes water molecules to polarize around the counterions, has also been considered. The folding energy of bound proteins has been seen to decrease proportionally with the increasing number of diffusion of counterions and their polarizability. Copyright © 2015 Elsevier B.V. All rights reserved.

Sculpting Mountains: Interactive Terrain Modeling Based on Subsurface Geology.

PubMed

Cordonnier, Guillaume; Cani, Marie-Paule; Benes, Bedrich; Braun, Jean; Galin, Eric

2018-05-01

Most mountain ranges are formed by the compression and folding of colliding tectonic plates. Subduction of one plate causes large-scale asymmetry while their layered composition (or stratigraphy) explains the multi-scale folded strata observed on real terrains. We introduce a novel interactive modeling technique to generate visually plausible, large scale terrains that capture these phenomena. Our method draws on both geological knowledge for consistency and on sculpting systems for user interaction. The user is provided hands-on control on the shape and motion of tectonic plates, represented using a new geologically-inspired model for the Earth crust. The model captures their volume preserving and complex folding behaviors under collision, causing mountains to grow. It generates a volumetric uplift map representing the growth rate of subsurface layers. Erosion and uplift movement are jointly simulated to generate the terrain. The stratigraphy allows us to render folded strata on eroded cliffs. We validated the usability of our sculpting interface through a user study, and compare the visual consistency of the earth crust model with geological simulation results and real terrains.

An immersed-boundary method for flow–structure interaction in biological systems with application to phonation

PubMed Central

Luo, Haoxiang; Mittal, Rajat; Zheng, Xudong; Bielamowicz, Steven A.; Walsh, Raymond J.; Hahn, James K.

2008-01-01

A new numerical approach for modeling a class of flow–structure interaction problems typically encountered in biological systems is presented. In this approach, a previously developed, sharp-interface, immersed-boundary method for incompressible flows is used to model the fluid flow and a new, sharp-interface Cartesian grid, immersed boundary method is devised to solve the equations of linear viscoelasticity that governs the solid. The two solvers are coupled to model flow–structure interaction. This coupled solver has the advantage of simple grid generation and efficient computation on simple, single-block structured grids. The accuracy of the solid-mechanics solver is examined by applying it to a canonical problem. The solution methodology is then applied to the problem of laryngeal aerodynamics and vocal fold vibration during human phonation. This includes a three-dimensional eigen analysis for a multi-layered vocal fold prototype as well as two-dimensional, flow-induced vocal fold vibration in a modeled larynx. Several salient features of the aerodynamics as well as vocal-fold dynamics are presented. PMID:19936017

RNA Tertiary Interactions in a Riboswitch Stabilize the Structure of a Kink Turn

PubMed Central

Schroeder, Kersten T.; Daldrop, Peter; Lilley, David M.J.

2011-01-01

Summary The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure. PMID:21893284

CYP3A4 substrate selection and substitution in the prediction of potential drug-drug interactions.

PubMed

Galetin, Aleksandra; Ito, Kiyomi; Hallifax, David; Houston, J Brian

2005-07-01

The complexity of in vitro kinetic phenomena observed for CYP3A4 substrates (homo- or heterotropic cooperativity) confounds the prediction of drug-drug interactions, and an evaluation of alternative and/or pragmatic approaches and substrates is needed. The current study focused on the utility of the three most commonly used CYP3A4 in vitro probes for the prediction of 26 reported in vivo interactions with azole inhibitors (increase in area under the curve ranged from 1.2 to 24, 50% in the range of potent inhibition). In addition to midazolam, testosterone, and nifedipine, quinidine was explored as a more "pragmatic" substrate due to its kinetic properties and specificity toward CYP3A4 in comparison with CYP3A5. Ki estimates obtained in human liver microsomes under standardized in vitro conditions for each of the four probes were used to determine the validity of substrate substitution in CYP3A4 drug-drug interaction prediction. Detailed inhibitor-related (microsomal binding, depletion over incubation time) and substrate-related factors (cooperativity, contribution of other metabolic pathways, or renal excretion) were incorporated in the assessment of the interaction potential. All four CYP3A4 probes predicted 69 to 81% of the interactions with azoles within 2-fold of the mean in vivo value. Comparison of simple and multisite mechanistic models and interaction prediction accuracy for each of the in vitro probes indicated that midazolam and quinidine in vitro data provided the best assessment of a potential interaction, with the lowest bias and the highest precision of the prediction. Further investigations with a wider range of inhibitors are required to substantiate these findings.

Lattice model simulation of interchain protein interactions and the folding dynamics and dimerization of the GCN4 Leucine zipper

NASA Astrophysics Data System (ADS)

Liu, Yanxin; Chapagain, Prem P.; Parra, Jose L.; Gerstman, Bernard S.

2008-01-01

The highest level in the hierarchy of protein structure and folding is the formation of protein complexes through protein-protein interactions. We have made modifications to a well established computer lattice model to expand its applicability to two-protein dimerization and aggregation. Based on Brownian dynamics, we implement translation and rotation moves of two peptide chains relative to each other, in addition to the intrachain motions already present in the model. We use this two-chain model to study the folding dynamics of the yeast transcription factor GCN4 leucine zipper. The calculated heat capacity curves agree well with experimental measurements. Free energy landscapes and median first passage times for the folding process are calculated and elucidate experimentally measured characteristics such as the multistate nature of the dimerization process.

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

PubMed Central

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

2015-01-01

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

Fluid-Structure Interactions as Flow Propagates Tangentially Over a Flexible Plate with Application to Voiced Speech Production

NASA Astrophysics Data System (ADS)

Westervelt, Andrea; Erath, Byron

2013-11-01

Voiced speech is produced by fluid-structure interactions that drive vocal fold motion. Viscous flow features influence the pressure in the gap between the vocal folds (i.e. glottis), thereby altering vocal fold dynamics and the sound that is produced. During the closing phases of the phonatory cycle, vortices form as a result of flow separation as air passes through the divergent glottis. It is hypothesized that the reduced pressure within a vortex core will alter the pressure distribution along the vocal fold surface, thereby aiding in vocal fold closure. The objective of this study is to determine the impact of intraglottal vortices on the fluid-structure interactions of voiced speech by investigating how the dynamics of a flexible plate are influenced by a vortex ring passing tangentially over it. A flexible plate, which models the medial vocal fold surface, is placed in a water-filled tank and positioned parallel to the exit of a vortex generator. The physical parameters of plate stiffness and vortex circulation are scaled with physiological values. As vortices propagate over the plate, particle image velocimetry measurements are captured to analyze the energy exchange between the fluid and flexible plate. The investigations are performed over a range of vortex formation numbers, and lateral displacements of the plate from the centerline of the vortex trajectory. Observations show plate oscillations with displacements directly correlated with the vortex core location.

Quantification of fold growth of frontal antiforms in the Zagros fold and thrust belt (Kurdistan, NE Iraq)

NASA Astrophysics Data System (ADS)

Bretis, Bernhard; Bartl, Nikolaus; Graseman, Bernhard; Lockhart, Duncan

2010-05-01

The Zagros fold and thrust belt is a seismically active orogen, where actual kinematic models based on GPS networks suggest a north-south shortening between Arabian and Eurasian in the order of 1.5-2.5 cm/yr. Most of this deformation is partitioned in south-southwest oriented folding and thrusting with northwest-southeast to north-south trending dextral strike slip faults. The Zagros fold and thrust belt is of great economic interest because it has been estimated that this area contains about 15% of the global recoverable hydrocarbons. Whereas the SE parts of the Zagros have been investigated by detailed geological studies, the NW extent being part of the Republic of Iraq have experienced considerably less attention. In this study we combine field work and remote sensing techniques in order to investigate the interaction of erosion and fold growth in the area NE of Erbil (Kurdistan, Iraq). In particular we focus on the interaction of the transient development of drainage patterns along growing antiforms, which directly reflects the kinematics of progressive fold growth. Detailed geomorphological studies of the Bana Bawi-, Permam- and Safeen fold trains show that these anticlines have not developed from subcylindrical embryonic folds but they have merged from different fold segments that joined laterally during fold amplification. This fold segments with length between 5 and 25 km have been detected by mapping ancient and modern river courses that initially cut the nose of growing folds and eventually got defeated leaving behind a wind gap. Fold segments, propagating in different directions force rivers to join resulting in steep gorges, which dissect the merging fold noses. Along rapidly lateral growing folds (e.g. at the SE end of the Bana Bawi Anticline) we observed "curved wind gaps", a new type of abandoned river course, where form of the wind gap mimics a formed nose of a growing antiform. The inherited curved segments of uplifted curved river courses strongly influence the development of the drainage system. This new model helps to detect embryonic fold segments of subcylindrical folds, which are otherwise difficult to identify.

Tunable electromagnetically induced transparency in coupled three-dimensional split-ring-resonator metamaterials

PubMed Central

Han, Song; Cong, Longqing; Lin, Hai; Xiao, Boxun; Yang, Helin; Singh, Ranjan

2016-01-01

Metamaterials have recently enabled coupling induced transparency due to interference effects in coupled subwavelength resonators. In this work, we present a three dimensional (3-D) metamaterial design with six-fold rotational symmetry that shows electromagnetically induced transparency with a strong polarization dependence to the incident electromagnetic wave due to the ultra-sharp resonance line width as a result of interaction between the constituent meta-atoms. However, when the six-fold rotationally symmetric unit cell design was re-arranged into a fourfold rotational symmetry, we observed the excitation of a polarization insensitive dual-band transparency. Thus, the 3-D split-ring resonators allow new schemes to observe single and multi-band classical analogues of electromagnetically induced transparencies that has huge potential applications in slowing down light, sensing modalities, and filtering functionalities either in the passive mode or the active mode where such effects could be tuned by integrating materials with dynamic properties. PMID:26857034

Neotectonics and structure of the Himalayan deformation front in the Kashmir Himalaya, India: Implication in defining what controls a blind thrust front in an active fold-thrust belt

NASA Astrophysics Data System (ADS)

Gavillot, Y. G.; Meigs, A.; Yule, J. D.; Rittenour, T. M.; Malik, M. O. A.

2014-12-01

Active tectonics of a deformation front constrains the kinematic evolution and structural interaction between the fold-thrust belt and most-recently accreted foreland basin. In Kashmir, the Himalayan Frontal thrust (HFT) is blind, characterized by a broad fold, the Suruin-Mastargh anticline (SMA), and displays no emergent faults cutting either limb. A lack of knowledge of the rate of shortening and structural framework of the SMA hampers quantifying the earthquake potential for the deformation front. Our study utilized the geomorphic expression of dated deformed terraces on the Ujh River in Kashmir. Six terraces are recognized, and three yield OSL ages of 53 ka, 33 ka, and 0.4 ka. Vector fold restoration of long terrace profiles indicates a deformation pattern characterized by regional uplift across the anticlinal axis and back-limb, and by fold limb rotation on the forelimb. Differential uplift across the fold trace suggests localized deformation. Dip data and stratigraphic thicknesses suggest that a duplex structure is emplaced at depth along the basal décollement, folding the overlying roof thrust and Siwalik-Muree strata into a detachment-like fold. Localized faulting at the fold axis explains the asymmetrical fold geometry. Folding of the oldest dated terrace, suggest that rock uplift rates across the SMA range between 2.0-1.8 mm/yr. Assuming a 25° dipping ramp for the blind structure on the basis of dip data constraints, the shortening rate across the SMA ranges between 4.4-3.8 mm/yr since ~53 ka. Of that rate, ~1 mm/yr is likely absorbed by minor faulting in the near field of the fold axis. Given that Himalaya-India convergence is ~18.8-11 mm/yr, internal faults north of the deformation front, such as the Riasi thrust absorbs more of the Himalayan shortening than does the HFT in Kashmir. We attribute a non-emergent thrust at the deformation front to reflect deformation controlled by pre-existing basin architecture in Kashmir, in which the thick succession of foreland strata Murree-Siwalik (8-9 km) overlie a deepened basal décollement. Blind thrusting reflects some combination of layer-parallel shortening, high stratigraphic overburden, relative youth of the HFT, and/or sustained low shortening rate on 10^5 yrs to longer timescales.

Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

PubMed Central

Snow, Christopher D.; Qiu, Linlin; Du, Deguo; Gai, Feng; Hagen, Stephen J.; Pande, Vijay S.

2004-01-01

We studied the microsecond folding dynamics of three β hairpins (Trp zippers 1–3, TZ1–TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLSaa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1–TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations. PMID:15020773

Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

NASA Astrophysics Data System (ADS)

Snow, Christopher D.; Qiu, Linlin; Du, Deguo; Gai, Feng; Hagen, Stephen J.; Pande, Vijay S.

2004-03-01

We studied the microsecond folding dynamics of three hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLSaa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.

A stoichiometry driven universal spatial organization of backbones of folded proteins: are there Chargaff's rules for protein folding?

PubMed

Mittal, A; Jayaram, B; Shenoy, Sandhya; Bawa, Tejdeep Singh

2010-10-01

Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that "preferential interactions" between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.

General mechanism of two-state protein folding kinetics.

PubMed

Rollins, Geoffrey C; Dill, Ken A

2014-08-13

We describe here a general model of the kinetic mechanism of protein folding. In the Foldon Funnel Model, proteins fold in units of secondary structures, which form sequentially along the folding pathway, stabilized by tertiary interactions. The model predicts that the free energy landscape has a volcano shape, rather than a simple funnel, that folding is two-state (single-exponential) when secondary structures are intrinsically unstable, and that each structure along the folding path is a transition state for the previous structure. It shows how sequential pathways are consistent with multiple stochastic routes on funnel landscapes, and it gives good agreement with the 9 order of magnitude dependence of folding rates on protein size for a set of 93 proteins, at the same time it is consistent with the near independence of folding equilibrium constant on size. This model gives estimates of folding rates of proteomes, leading to a median folding time in Escherichia coli of about 5 s.

Controlling electric, magnetic, and chiral dipolar emission with PT-symmetric potentials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alaeian, Hadiseh; Dionne, Jennifer A.

We investigate the effect of parity-time (PT) symmetric optical potentials on the radiation of achiral and chiral dipole sources. Two properties unique to PT-symmetric potentials are observed. First, the dipole can be tuned to behave as a strong optical emitter or absorber based on the non-Hermiticity parameter and the dipole location. Second, exceptional points give rise to new system resonances that lead to orders-of-magnitude enhancements in the dipolar emitted or absorbed power. Utilizing these properties, we show that enantiomers of chiral molecules near PT-symmetric metamaterials exhibit a 4.5-fold difference in their emitted power and decay rate. The results of thismore » work could enable new atom-cavity interactions for quantum optics, as well as all-optical enantioselective separation.« less

Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins.

PubMed

Solis, Armando D

2015-12-01

To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20-letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long-range (contact) interactions among amino acids in natively-folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well-defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well-known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long-range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches-including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs-fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. © 2015 Wiley Periodicals, Inc.

Electrostatic lipid-protein interactions sequester the curli amyloid fold on the lipopolysaccharide membrane surface.

PubMed

Swasthi, Hema M; Mukhopadhyay, Samrat

2017-12-01

Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein-protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Salt-bridge networks within globular and disordered proteins: characterizing trends for designable interactions.

PubMed

Basu, Sankar; Mukharjee, Debasish

2017-07-01

There has been considerable debate about the contribution of salt bridges to the stabilization of protein folds, in spite of their participation in crucial protein functions. Salt bridges appear to contribute to the activity-stability trade-off within proteins by bringing high-entropy charged amino acids into close contacts during the course of their functions. The current study analyzes the modes of association of salt bridges (in terms of networks) within globular proteins and at protein-protein interfaces. While the most common and trivial type of salt bridge is the isolated salt bridge, bifurcated salt bridge appears to be a distinct salt-bridge motif having a special topology and geometry. Bifurcated salt bridges are found ubiquitously in proteins and interprotein complexes. Interesting and attractive examples presenting different modes of interaction are highlighted. Bifurcated salt bridges appear to function as molecular clips that are used to stitch together large surface contours at interacting protein interfaces. The present work also emphasizes the key role of salt-bridge-mediated interactions in the partial folding of proteins containing long stretches of disordered regions. Salt-bridge-mediated interactions seem to be pivotal to the promotion of "disorder-to-order" transitions in small disordered protein fragments and their stabilization upon binding. The results obtained in this work should help to guide efforts to elucidate the modus operandi of these partially disordered proteins, and to conceptualize how these proteins manage to maintain the required amount of disorder even in their bound forms. This work could also potentially facilitate explorations of geometrically specific designable salt bridges through the characterization of composite salt-bridge networks. Graphical abstract ᅟ.

H-Bond Self-Assembly: Folding versus Duplex Formation.

PubMed

Núñez-Villanueva, Diego; Iadevaia, Giulia; Stross, Alexander E; Jinks, Michael A; Swain, Jonathan A; Hunter, Christopher A

2017-05-17

Linear oligomers equipped with complementary H-bond donor (D) and acceptor (A) sites can interact via intermolecular H-bonds to form duplexes or fold via intramolecular H-bonds. These competing equilibria have been quantified using NMR titration and dilution experiments for seven systems featuring different recognition sites and backbones. For all seven architectures, duplex formation is observed for homo-sequence 2-mers (AA·DD) where there are no competing folding equilibria. The corresponding hetero-sequence AD 2-mers also form duplexes, but the observed self-association constants are strongly affected by folding equilibria in the monomeric states. When the backbone is flexible (five or more rotatable bonds separating the recognition sites), intramolecular H-bonding is favored, and the folded state is highly populated. For these systems, the stability of the AD·AD duplex is 1-2 orders of magnitude lower than that of the corresponding AA·DD duplex. However, for three architectures which have more rigid backbones (fewer than five rotatable bonds), intramolecular interactions are not observed, and folding does not compete with duplex formation. These systems are promising candidates for the development of longer, mixed-sequence synthetic information molecules that show sequence-selective duplex formation.

Probabilistic analysis for identifying the driving force of protein folding

NASA Astrophysics Data System (ADS)

Tokunaga, Yoshihiko; Yamamori, Yu; Matubayasi, Nobuyuki

2018-03-01

Toward identifying the driving force of protein folding, energetics was analyzed in water for Trp-cage (20 residues), protein G (56 residues), and ubiquitin (76 residues) at their native (folded) and heat-denatured (unfolded) states. All-atom molecular dynamics simulation was conducted, and the hydration effect was quantified by the solvation free energy. The free-energy calculation was done by employing the solution theory in the energy representation, and it was seen that the sum of the protein intramolecular (structural) energy and the solvation free energy is more favorable for a folded structure than for an unfolded one generated by heat. Probabilistic arguments were then developed to determine which of the electrostatic, van der Waals, and excluded-volume components of the interactions in the protein-water system governs the relative stabilities between the folded and unfolded structures. It was found that the electrostatic interaction does not correspond to the preference order of the two structures. The van der Waals and excluded-volume components were shown, on the other hand, to provide the right order of preference at probabilities of almost unity, and it is argued that a useful modeling of protein folding is possible on the basis of the excluded-volume effect.

A molecular mechanism of chaperone-client recognition

PubMed Central

He, Lichun; Sharpe, Timothy; Mazur, Adam; Hiller, Sebastian

2016-01-01

Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature. PMID:28138538

UFO (UnFold Operator) computer program abstract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kissel, L.; Biggs, F.

UFO (UnFold Operator) is an interactive user-oriented computer program designed to solve a wide range of problems commonly encountered in physical measurements. This document provides a summary of the capabilities of version 3A of UFO.

Unsteady flow motions in the supraglottal region during phonation

NASA Astrophysics Data System (ADS)

Luo, Haoxiang; Dai, Hu

2008-11-01

The highly unsteady flow motions in the larynx are not only responsible for producing the fundamental frequency tone in phonation, but also have a significant contribution to the broadband noise in the human voice. In this work, the laryngeal flow is modeled either as an incompressible pulsatile jet confined in a two-dimensional channel, or a pressure-driven flow modulated by a pair of viscoelastic vocal folds through the flow--structure interaction. The flow in the supraglottal region is found to be dominated by large-scale vortices whose unsteady motions significantly deflect the glottal jet. In the flow--structure interaction, a hybrid model based on the immersed-boundary method is developed to simulate the flow-induced vocal fold vibration, which involves a three-dimensional vocal fold prototype and a two-dimensional viscous flow. Both the flow behavior and the vibratory characteristics of the vocal folds will be presented.

Stable chromosome condensation revealed by chromosome conformation capture

PubMed Central

Eagen, Kyle P.; Hartl, Tom A.; Kornberg, Roger D.

2015-01-01

SUMMARY Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to ten-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes. PMID:26544940

High conductance values in π-folded molecular junctions

NASA Astrophysics Data System (ADS)

Carini, Marco; Ruiz, Marta P.; Usabiaga, Imanol; Fernández, José A.; Cocinero, Emilio J.; Melle-Franco, Manuel; Diez-Perez, Ismael; Mateo-Alonso, Aurelio

2017-05-01

Folding processes play a crucial role in the development of function in biomacromolecules. Recreating this feature on synthetic systems would not only allow understanding and reproducing biological functions but also developing new functions. This has inspired the development of conformationally ordered synthetic oligomers known as foldamers. Herein, a new family of foldamers, consisting of an increasing number of anthracene units that adopt a folded sigmoidal conformation by a combination of intramolecular hydrogen bonds and aromatic interactions, is reported. Such folding process opens up an efficient through-space charge transport channel across the interacting anthracene moieties. In fact, single-molecule conductance measurements carried out on this series of foldamers, using the scanning tunnelling microscopy-based break-junction technique, reveal exceptionally high conductance values in the order of 10-1 G0 and a low length decay constant of 0.02 Å-1 that exceed the values observed in molecular junctions that make use of through-space charge transport pathways.

Peptide folding in the presence of interacting protein crowders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bille, Anna, E-mail: anna.bille@thep.lu.se; Irbäck, Anders, E-mail: anders@thep.lu.se; Mohanty, Sandipan, E-mail: s.mohanty@fz-juelich.de

2016-05-07

Using Monte Carlo methods, we explore and compare the effects of two protein crowders, BPTI and GB1, on the folding thermodynamics of two peptides, the compact helical trp-cage and the β-hairpin-forming GB1m3. The thermally highly stable crowder proteins are modeled using a fixed backbone and rotatable side-chains, whereas the peptides are free to fold and unfold. In the simulations, the crowder proteins tend to distort the trp-cage fold, while having a stabilizing effect on GB1m3. The extent of the effects on a given peptide depends on the crowder type. Due to a sticky patch on its surface, BPTI causes largermore » changes than GB1 in the melting properties of the peptides. The observed effects on the peptides stem largely from attractive and specific interactions with the crowder surfaces, and differ from those seen in reference simulations with purely steric crowder particles.« less

Topological frustration in βα-repeat proteins: sequence diversity modulates the conserved folding mechanisms of α/β/α sandwich proteins

PubMed Central

Hills, Ronald D.; Kathuria, Sagar V.; Wallace, Louise A.; Day, Iain J.; Brooks, Charles L.; Matthews, C. Robert

2010-01-01

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of non-local interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα–repeat proteins, which are dominated by local interactions, a combined experimental and simulation analysis was performed on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured sub-millisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine and valine (ILV) side-chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3 and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side-chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely-folded unproductive intermediates are a common property of the βα-repeat motif. PMID:20226790

Response of lead-acid batteries to chopper-controlled discharge: Preliminary results

NASA Technical Reports Server (NTRS)

Cataldo, R. L.

1978-01-01

The preliminary results of simulated electric vehicle, chopper, speed controller discharge of a battery show energy output losses up to 25 percent compared to constant current discharges at the same average discharge current of 100 amperes. These energy losses are manifested as temperature rises during discharge, amounting to a two-fold increase for a 400-ampere pulse compared to the constant current case. Because of the potentially large energy inefficiency, the results suggest that electric vehicle battery/speed controller interaction must be carefully considered in vehicle design.

Response of lead-acid batteries to chopper-controlled discharge

NASA Technical Reports Server (NTRS)

Cataldo, R. L.

1978-01-01

The preliminary results of simulated electric vehicle, chopper, speed controller discharge of a battery show energy output losses at up to 25 percent compared to constant current discharges at the same average discharge current of 100 A. These energy losses are manifested as temperature rises during discharge, amounting to a two-fold increase for a 400-A pulse compared to the constant current case. Because of the potentially large energy inefficiency, the results suggest that electric vehicle battery/speed controller interaction must be carefully considered in vehicle design.

Interaction of β-sheet folds with a gold surface.

PubMed

Hoefling, Martin; Monti, Susanna; Corni, Stefano; Gottschalk, Kay Eberhard

2011-01-01

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance.

Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix

PubMed Central

De, Soumya; Okon, Mark; Graves, Barbara J.; McIntosh, Lawrence P.

2017-01-01

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge–dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity. PMID:26920109

Protein folding: complex potential for the driving force in a two-dimensional space of collective variables.

PubMed

Chekmarev, Sergei F

2013-10-14

Using the Helmholtz decomposition of the vector field of folding fluxes in a two-dimensional space of collective variables, a potential of the driving force for protein folding is introduced. The potential has two components. One component is responsible for the source and sink of the folding flows, which represent respectively, the unfolded states and the native state of the protein, and the other, which accounts for the flow vorticity inherently generated at the periphery of the flow field, is responsible for the canalization of the flow between the source and sink. The theoretical consideration is illustrated by calculations for a model β-hairpin protein.

Influence of the ventricular folds on a voice source with specified vocal fold motion1

PubMed Central

McGowan, Richard S.; Howe, Michael S.

2010-01-01

The unsteady drag on the vocal folds is the major source of sound during voiced speech. The drag force is caused by vortex shedding from the vocal folds. The influence of the ventricular folds (i.e., the “false” vocal folds that protrude into the vocal tract a short distance downstream of the glottis) on the drag and the voice source are examined in this paper by means of a theoretical model involving vortex sheets in a two-dimensional geometry. The effect of the ventricular folds on the output acoustic pressure is found to be small when the movement of the vocal folds is prescribed. It is argued that the effect remains small when fluid-structure interactions account for vocal fold movement. These conclusions can be justified mathematically when the characteristic time scale for change in the velocity of the glottal jet is large compared to the time it takes for a vortex disturbance to be convected through the vocal fold and ventricular fold region. PMID:20329852

Enhancement of cell recognition in vitro by dual-ligand cancer targeting gold naoparticles

PubMed Central

Li, Xi; Zhou, Hongyu; Yang, Lei; Du, Guoqing; Pai-Panandiker, Atmaram; Huang, Xuefei; Yan, Bing

2011-01-01

A dual-ligand gold nanoparticle (DLGNP) was designed and synthesized to explore the therapeutic benefits of multivalent interactions between gold nanoparticles (GNPs) and cancer cells. DLGNP was tested on human epidermal cancer cells (KB), which had high expression of folate receptor. The cellular uptake of DLGNP was increased by 3.9 and 12.7 folds compared with GNP-folate or GNP-glucose. The enhanced cell recognition was due to multivalent interactions between both ligands on GNPs and cancer cells as shown by the ligand competition experiments. Furthermore, the multivalent interactions increased contrast between cells with high and low expression of folate receptors. The enhanced cell recognition enabled DLGNP to kill KB cells under X-ray irradiation at a dose that was safe to folate receptor low-expression (such as normal) cells. Thus DLGP has the potential to be a cancer-specific nano-theranostic agent. PMID:21232787

Targeting diseased tissues by pHLIP insertion at low cell surface pH.

PubMed

Andreev, Oleg A; Engelman, Donald M; Reshetnyak, Yana K

2014-01-01

The discovery of the pH Low Insertion Peptides (pHLIPs®) provides an opportunity to develop imaging and drug delivery agents targeting extracellular acidity. Extracellular acidity is associated with many pathological states, such as those in cancer, ischemic stroke, neurotrauma, infection, lacerations, and others. The metabolism of cells in injured or diseased tissues often results in the acidification of the extracellular environment, so acidosis might be useful as a general marker for the imaging and treatment of diseased states if an effective targeting method can be developed. The molecular mechanism of a pHLIP peptide is based on pH-dependent membrane-associated folding. pHLIPs, being moderately hydrophobic peptides, have high affinities for cellular membranes at normal pH, but fold and insert across membranes at low pH, allowing them to sense pH at the surfaces of cells in diseased tissues, where it is the lowest. Here we discuss the main principles of pHLIP interactions with membrane lipid bilayers at neutral and low pHs, the possibility of tuning the folding and insertion pH by peptide sequence variation, and potential applications of pHLIPs for imaging, therapy and image-guided interventions.

Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.

PubMed

Arpino, James A J; Reddington, Samuel C; Halliwell, Lisa M; Rizkallah, Pierre J; Jones, D Dafydd

2014-06-10

Altering a protein's backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFP(G4Δ)) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFP(G4Δ) retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFP(G4Δ) structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Development of Pyrazolone and Isoxazol-5-one Cambinol Analogues as Sirtuin Inhibitors

PubMed Central

2015-01-01

Sirtuins are a family of NAD+-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition. PMID:24697269

Single Folding Optical Potential for Elastic Scattering of Protons from 14N and 16O in a Wide Range of Energies

NASA Astrophysics Data System (ADS)

Hamada, Sh.

2018-03-01

Available experimental data for protons elastically scattered from 14N and 16O target nuclei are reanalyzed within the framework of single folding optical potential (SFOP) model. In this model, the real part of the potential is derived on the basis of single folding potential. The renormalization factor N r is extracted for the two aforementioned nuclear systems. Theoretical calculations fairly reproduce the experimental data in the whole angular range. Energy dependence of real and imaginary volume integrals as well as reaction cross sections are discussed.

Design of HIV-1-PR inhibitors that do not create resistance: blocking the folding of single monomers.

PubMed

Broglia, Ricardo A; Tiana, Guido; Sutto, Ludovico; Provasi, Davide; Simona, Fabio

2005-10-01

The main problems found in designing drugs are those of optimizing the drug-target interaction and of avoiding the insurgence of resistance. We suggest a scheme for the design of inhibitors that can be used as leads for the development of a drug and that do not face either of these problems, and then apply it to the case of HIV-1-PR. It is based on the knowledge that the folding of single-domain proteins, such as each of the monomers forming the HIV-1-PR homodimer, is controlled by local elementary structures (LES), stabilized by local contacts among hydrophobic, strongly interacting, and highly conserved amino acids that play a central role in the folding process. Because LES have evolved over many generations to recognize and strongly interact with each other so as to make the protein fold fast and avoid aggregation with other proteins, highly specific (and thus little toxic) as well as effective folding-inhibitor molecules suggest themselves: short peptides (or eventually their mimetic molecules) displaying the same amino acid sequence of that of LES (p-LES). Aside from being specific and efficient, these inhibitors are expected not to induce resistance; in fact, mutations in HIV-1-PR that successfully avoid the action of p-LES imply the destabilization of one or more LES and thus should lead to protein denaturation. Making use of Monte Carlo simulations, we first identify the LES of the HIV-1-PR and then show that the corresponding p-LES peptides act as effective inhibitors of the folding of the protease.

Minimization and Optimization of Designed β-Hairpin Folds

PubMed Central

Andersen, Niels H.; Olsen, Katherine A.; Fesinmeyer, R. Matthew; Tan, Xu; Hudson, F. Michael; Eidenschink, Lisa A.; Farazi, Shabnam R.

2011-01-01

Mimimized β hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, ΔGU298 ≥ +7 kJ/mol) and 12-residue (KTWNPATGKWTE, ΔGU298 = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-π interaction between Lys1 and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hε3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the –TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small ΔCp for unfolding (+420 JK−1/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH2) with the fold stability reduced to ΔGU278 = −0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also forms the same hairpin-stabilizing W/W interaction. PMID:16669679

Engineering Aromatic-Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins.

PubMed

Balakrishnan, Swati; Sarma, Siddhartha P

2017-08-22

Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b 5 . Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b 5 . The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a C m higher than that of apo-cytochrome b 5 and are more stable to chaotropic agents than apo-cytochrome b 5 . The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.

A theoretical study of F0-F1 interaction with application to resonant speaking and singing voice.

PubMed

Titze, Ingo R

2004-09-01

An interactive source-filter system, consisting of a three-mass body-cover model of the vocal folds and a wave reflection model of the vocal tract, was used to test the dependence of vocal fold vibration on the vocal tract. The degree of interaction is governed by the epilarynx tube, which raises the vocal tract impedance to match the impedance of the glottis. The key component of the impedance is inertive reactance. Whenever there is inertive reactance, the vocal tract assists the vocal folds in vibration. The amplitude of vibration and the glottal flow can more than double, and the oral radiated power can increase up to 10 dB. As F0 approaches F1, the first formant frequency, the interactive source-filter system loses its advantage (because inertive reactance changes to compliant reactance) and the noninteractive system produces greater vocal output. Thus, from a voice training and control standpoint, there may be reasons to operate the system in either interactive and noninteractive modes. The harmonics 2F0 and 3F0 can also benefit from being positioned slightly below F1.

Stereochemistry and solvent role in protein folding: nuclear magnetic resonance and molecular dynamics studies of poly-L and alternating-L,D homopolypeptides in dimethyl sulfoxide.

PubMed

Srivastava, Kinshuk Raj; Kumar, Anil; Goyal, Bhupesh; Durani, Susheel

2011-05-26

The competing interactions folding and unfolding protein structure remain obscure. Using homopolypeptides, we ask if poly-L structure may have a role. We mutate the structure to alternating-L,D stereochemistry and substitute water as the fold-promoting solvent with methanol and dimethyl sulfoxide (DMSO) as the fold-denaturing solvents. Circular dichroism and molecular dynamics established previously that, while both isomers were folded in water, the poly-L isomer was unfolded and alternating-L,D isomer folded in methanol. Nuclear magnetic resonance and molecular dynamics establish now that both isomers are unfolded in DMSO. We calculated energetics of folding-unfolding equilibrium with water and methanol as solvents. We have now calculated interactions of unfolded polypeptide structures with DMSO as solvent. Methanol was found to unfold and water fold poly-L structure as a dielectric. DMSO has now been found to unfold both poly-L and alternating-L,D structures by strong solvation of peptides to disrupt their hydrogen bonds. Accordingly, we propose that while linked peptides fold protein structure with hydrogen bonds they unfold the structure electrostatically due to the stereochemical effect of the poly-L structure. Protein folding to ordering of peptide hydrogen bonds with water as canonical solvent may thus involve two specific and independent solvent effects-one, strong screening of electrostatics of poly-L linked peptides, and two, weak dipolar solvation of peptides. Correspondingly, protein denaturation may involve two independent solvent effects-one, weak dielectric to unfold poly-L structure electrostatically, and two, strong polarity to disrupt peptide hydrogen bonds by solvation of peptides.

Selective far-field addressing of coupled quantum dots in a plasmonic nanocavity.

PubMed

Tang, Jianwei; Xia, Juan; Fang, Maodong; Bao, Fanglin; Cao, Guanjun; Shen, Jianqi; Evans, Julian; He, Sailing

2018-04-27

Plasmon-emitter hybrid nanocavity systems exhibit strong plasmon-exciton interactions at the single-emitter level, showing great potential as testbeds and building blocks for quantum optics and informatics. However, reported experiments involve only one addressable emitting site, which limits their relevance for many fundamental questions and devices involving interactions among emitters. Here we open up this critical degree of freedom by demonstrating selective far-field excitation and detection of two coupled quantum dot emitters in a U-shaped gold nanostructure. The gold nanostructure functions as a nanocavity to enhance emitter interactions and a nanoantenna to make the emitters selectively excitable and detectable. When we selectively excite or detect either emitter, we observe photon emission predominantly from the target emitter with up to 132-fold Purcell-enhanced emission rate, indicating individual addressability and strong plasmon-exciton interactions. Our work represents a step towards a broad class of plasmonic devices that will enable faster, more compact optics, communication and computation.

Viroporins, Examples of the Two-Stage Membrane Protein Folding Model.

PubMed

Martinez-Gil, Luis; Mingarro, Ismael

2015-06-26

Viroporins are small, α-helical, hydrophobic virus encoded proteins, engineered to form homo-oligomeric hydrophilic pores in the host membrane. Viroporins participate in multiple steps of the viral life cycle, from entry to budding. As any other membrane protein, viroporins have to find the way to bury their hydrophobic regions into the lipid bilayer. Once within the membrane, the hydrophobic helices of viroporins interact with each other to form higher ordered structures required to correctly perform their porating activities. This two-step process resembles the two-stage model proposed for membrane protein folding by Engelman and Poppot. In this review we use the membrane protein folding model as a leading thread to analyze the mechanism and forces behind the membrane insertion and folding of viroporins. We start by describing the transmembrane segment architecture of viroporins, including the number and sequence characteristics of their membrane-spanning domains. Next, we connect the differences found among viroporin families to their viral genome organization, and finalize focusing on the pathways used by viroporins in their way to the membrane and on the transmembrane helix-helix interactions required to achieve proper folding and assembly.

Coarse Graining to Investigate Membrane Induced Peptide Folding of Anticancer Peptides

NASA Astrophysics Data System (ADS)

Ganesan, Sai; Xu, Hongcheng; Matysiak, Silvina

Information about membrane induced peptide folding mechanisms using all-atom molecular dynamics simulations is a challenge due to time and length scale issues.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.These two dummy particles represent a fluctuating dipole,thus introducing structural polarization into the coarse-grained model.With this model,we were able to achieve significant α- β secondary structure content de novo,without any added bias.We extended the model to zwitterionic and anionic lipids,by adding oppositely charged dummy particles inside polar beads, to capture the ability of the head group region to form hydrogen bonds.We use zwitterionic POPC and anionic POPS as our model lipids, and a cationic anticancer peptide,SVS1,as our model peptide.We have characterized the driving forces for SVS1 folding on lipid bilayers with varying anionic and zwitterionic lipid compositions.Based on our results, dipolar interactions between peptide backbone and lipid head groups contribute to stabilize folded conformations.Cooperativity in folding is induced by both intra peptide and membrane-peptide interaction.

Citronellal assumes a folded conformation in solution due to dispersion interactions: A joint NMR-DFT analysis

NASA Astrophysics Data System (ADS)

Nardini, Viviani; Dias, Luis Gustavo; Palaretti, Vinicius; da Silva, Gil Valdo José

2018-04-01

Citronellal, an acyclic monoterpenoid, is a small molecule suitable for systematic scanning of its conformational geometric parameters in solution or in the gas phase. We have studied the conformational distribution of citronellal by correlating its structure and theoretical chemical shifts with nuclear magnetic resonance data. Interestingly, folded conformations were the most relevant, as confirmed by NOE experiments. We concluded that the conformational distribution is due to intramolecular dispersion interactions.

Pharmacological chaperoning: a primer on mechanism and pharmacology.

PubMed

Leidenheimer, Nancy J; Ryder, Katelyn G

2014-05-01

Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast. This is most evident in the treatment of lysosomal storage disorders, cystic fibrosis, and nephrogenic diabetes insipidus, for which proof of principle in humans has been demonstrated. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of aerodynamic characteristics of a coupled fluid-structure system using generalized Bernoulli’s principle: An application to vocal folds vibration

PubMed Central

Zhang, Lucy T.; Yang, Jubiao

2017-01-01

In this work we explore the aerodynamics flow characteristics of a coupled fluid-structure interaction system using a generalized Bernoulli equation derived directly from the Cauchy momentum equations. Unlike the conventional Bernoulli equation where incompressible, inviscid, and steady flow conditions are assumed, this generalized Bernoulli equation includes the contributions from compressibility, viscous, and unsteadiness, which could be essential in defining aerodynamic characteristics. The application of the derived Bernoulli’s principle is on a fully-coupled fluid-structure interaction simulation of the vocal folds vibration. The coupled system is simulated using the immersed finite element method where compressible Navier-Stokes equations are used to describe the air and an elastic pliable structure to describe the vocal fold. The vibration of the vocal fold works to open and close the glottal flow. The aerodynamics flow characteristics are evaluated using the derived Bernoulli’s principles for a vibration cycle in a carefully partitioned control volume based on the moving structure. The results agree very well to experimental observations, which validate the strategy and its use in other types of flow characteristics that involve coupled fluid-structure interactions. PMID:29527541

Evaluation of aerodynamic characteristics of a coupled fluid-structure system using generalized Bernoulli's principle: An application to vocal folds vibration.

PubMed

Zhang, Lucy T; Yang, Jubiao

2016-12-01

In this work we explore the aerodynamics flow characteristics of a coupled fluid-structure interaction system using a generalized Bernoulli equation derived directly from the Cauchy momentum equations. Unlike the conventional Bernoulli equation where incompressible, inviscid, and steady flow conditions are assumed, this generalized Bernoulli equation includes the contributions from compressibility, viscous, and unsteadiness, which could be essential in defining aerodynamic characteristics. The application of the derived Bernoulli's principle is on a fully-coupled fluid-structure interaction simulation of the vocal folds vibration. The coupled system is simulated using the immersed finite element method where compressible Navier-Stokes equations are used to describe the air and an elastic pliable structure to describe the vocal fold. The vibration of the vocal fold works to open and close the glottal flow. The aerodynamics flow characteristics are evaluated using the derived Bernoulli's principles for a vibration cycle in a carefully partitioned control volume based on the moving structure. The results agree very well to experimental observations, which validate the strategy and its use in other types of flow characteristics that involve coupled fluid-structure interactions.

Systematic study on the isotopic behavior of fusion barrier using the density-dependent nucleon-nucleon interactions

NASA Astrophysics Data System (ADS)

Ghodsi, O. N.; Khalaj, M.

By changing the neutron and nuclear matter incompressibility constant K, we investigate the isotopic behavior of the fusion barriers for the collision of large number of different isotopes with condition of 0.7 ≤ N/Z ≤ 1.36. Here, the double folding (DF) model which is accompanied by density-dependent (DD) versions of M3Y interactions is adopted as a basic heavy ion-ion potential. We show that the selected DD potentials predict a linear behavior for the calculated fusion barrier heights as a function of (N/Z - 1) for both proton- and neutron-rich systems. Moreover, the results indicate that the isotopic behavior of these values depend linearly on the change in the K constant. The isotopic studies conducted on the fusion cross-section also shows that the properties of the nuclear matter in the range of energy which is below the fusion barrier will quite affect the fusion process.

Free energy of RNA-counterion interactions in a tight-binding model computed by a discrete space mapping

NASA Astrophysics Data System (ADS)

Henke, Paul S.; Mak, Chi H.

2014-08-01

The thermodynamic stability of a folded RNA is intricately tied to the counterions and the free energy of this interaction must be accounted for in any realistic RNA simulations. Extending a tight-binding model published previously, in this paper we investigate the fundamental structure of charges arising from the interaction between small functional RNA molecules and divalent ions such as Mg2+ that are especially conducive to stabilizing folded conformations. The characteristic nature of these charges is utilized to construct a discretely connected energy landscape that is then traversed via a novel application of a deterministic graph search technique. This search method can be incorporated into larger simulations of small RNA molecules and provides a fast and accurate way to calculate the free energy arising from the interactions between an RNA and divalent counterions. The utility of this algorithm is demonstrated within a fully atomistic Monte Carlo simulation of the P4-P6 domain of the Tetrahymena group I intron, in which it is shown that the counterion-mediated free energy conclusively directs folding into a compact structure.

Free energy of RNA-counterion interactions in a tight-binding model computed by a discrete space mapping.

PubMed

Henke, Paul S; Mak, Chi H

2014-08-14

The thermodynamic stability of a folded RNA is intricately tied to the counterions and the free energy of this interaction must be accounted for in any realistic RNA simulations. Extending a tight-binding model published previously, in this paper we investigate the fundamental structure of charges arising from the interaction between small functional RNA molecules and divalent ions such as Mg(2+) that are especially conducive to stabilizing folded conformations. The characteristic nature of these charges is utilized to construct a discretely connected energy landscape that is then traversed via a novel application of a deterministic graph search technique. This search method can be incorporated into larger simulations of small RNA molecules and provides a fast and accurate way to calculate the free energy arising from the interactions between an RNA and divalent counterions. The utility of this algorithm is demonstrated within a fully atomistic Monte Carlo simulation of the P4-P6 domain of the Tetrahymena group I intron, in which it is shown that the counterion-mediated free energy conclusively directs folding into a compact structure.

SimRNA: a coarse-grained method for RNA folding simulations and 3D structure prediction.

PubMed

Boniecki, Michal J; Lach, Grzegorz; Dawson, Wayne K; Tomala, Konrad; Lukasz, Pawel; Soltysinski, Tomasz; Rother, Kristian M; Bujnicki, Janusz M

2016-04-20

RNA molecules play fundamental roles in cellular processes. Their function and interactions with other biomolecules are dependent on the ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. Here, we present SimRNA: a new method for computational RNA 3D structure prediction, which uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information, and, on established test sequences, it recapitulates secondary structure with high accuracy, including correct prediction of pseudoknots. For modeling of complex 3D structures, it can use additional restraints, derived from experimental or computational analyses, including information about secondary structure and/or long-range contacts. SimRNA also can be used to analyze conformational landscapes and identify potential alternative structures. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance.

PubMed

Rajabi, Mohsen; Struble, Evi; Zhou, Zhaohua; Karnaukhova, Elena

2012-01-01

Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states. SCOPE OF RESEARCH: We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout. Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation. This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies. Published by Elsevier B.V.

General Mechanism of Two-State Protein Folding Kinetics

PubMed Central

Rollins, Geoffrey C.; Dill, Ken A.

2016-01-01

We describe here a general model of the kinetic mechanism of protein folding. In the Foldon Funnel Model, proteins fold in units of secondary structures, which form sequentially along the folding pathway, stabilized by tertiary interactions. The model predicts that the free energy landscape has a volcano shape, rather than a simple funnel, that folding is two-state (single-exponential) when secondary structures are intrinsically unstable, and that each structure along the folding path is a transition state for the previous structure. It shows how sequential pathways are consistent with multiple stochastic routes on funnel landscapes, and it gives good agreement with the 9 order of magnitude dependence of folding rates on protein size for a set of 93 proteins, at the same time it is consistent with the near independence of folding equilibrium constant on size. This model gives estimates of folding rates of proteomes, leading to a median folding time in Escherichia coli of about 5 s. PMID:25056406

Contribution to the Prediction of the Fold Code: Application to Immunoglobulin and Flavodoxin Cases

PubMed Central

Banach, Mateusz; Prudhomme, Nicolas; Carpentier, Mathilde; Duprat, Elodie; Papandreou, Nikolaos; Kalinowska, Barbara; Chomilier, Jacques; Roterman, Irena

2015-01-01

Background Folding nucleus of globular proteins formation starts by the mutual interaction of a group of hydrophobic amino acids whose close contacts allow subsequent formation and stability of the 3D structure. These early steps can be predicted by simulation of the folding process through a Monte Carlo (MC) coarse grain model in a discrete space. We previously defined MIRs (Most Interacting Residues), as the set of residues presenting a large number of non-covalent neighbour interactions during such simulation. MIRs are good candidates to define the minimal number of residues giving rise to a given fold instead of another one, although their proportion is rather high, typically [15-20]% of the sequences. Having in mind experiments with two sequences of very high levels of sequence identity (up to 90%) but different folds, we combined the MIR method, which takes sequence as single input, with the “fuzzy oil drop” (FOD) model that requires a 3D structure, in order to estimate the residues coding for the fold. FOD assumes that a globular protein follows an idealised 3D Gaussian distribution of hydrophobicity density, with the maximum in the centre and minima at the surface of the “drop”. If the actual local density of hydrophobicity around a given amino acid is as high as the ideal one, then this amino acid is assigned to the core of the globular protein, and it is assumed to follow the FOD model. Therefore one obtains a distribution of the amino acids of a protein according to their agreement or rejection with the FOD model. Results We compared and combined MIR and FOD methods to define the minimal nucleus, or keystone, of two populated folds: immunoglobulin-like (Ig) and flavodoxins (Flav). The combination of these two approaches defines some positions both predicted as a MIR and assigned as accordant with the FOD model. It is shown here that for these two folds, the intersection of the predicted sets of residues significantly differs from random selection. It reduces the number of selected residues by each individual method and allows a reasonable agreement with experimentally determined key residues coding for the particular fold. In addition, the intersection of the two methods significantly increases the specificity of the prediction, providing a robust set of residues that constitute the folding nucleus. PMID:25915049

Non-covalent conjugation of cutinase from Fusarium sp. ICT SAC1 with pectin for enhanced stability: Process minutiae, kinetics, thermodynamics and structural study.

PubMed

Muley, Abhijeet B; Chaudhari, Sandeep A; Singhal, Rekha S

2017-09-01

Cutinase, a member of α/β-fold hydrolase family possess potentially diverse applications in several industrial processes and products. The present work aims towards thermo-stabilization of cutinase from novel source Fusarium sp. ICT SAC1 via non-covalent interaction with polysaccharides. Although all six polysaccharides chosen for study enhanced the thermal stability, pectin was found to be most promising. The interaction protocol for cutinase with pectin was optimized sequentially with respect to the ratio of enzyme to pectin, solution pH, and buffer strength. Cutinase-pectin conjugate under optimized conditions (1:12, pH-6.5, 50mM) showed enhanced thermal stability as evident from lower inactivation rate constant, higher half-life and D-value within the 40-55°C. A slender rise in K m and V max values and enhanced thermodynamic parameters of cutinase-pectin conjugate were observed after non-covalent interaction. Entropy values were 1.5-fold higher for cutinase-pectin conjugate at each temperature suggesting an upsurge in number of protein molecules in a transition activated state. Positive values of entropy for both forms of cutinase suggested a rise in disordered conformation. Noticeable conformational changes in cutinase after conjugation with pectin were confirmed by FTIR as well as fluorescence emission spectra. An increment in helix to turn conversion was observed in complexed cutinase vis-à-vis free cutinase. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure-function correlations of pulmonary surfactant protein SP-B and the saposin-like family of proteins.

PubMed

Olmeda, Bárbara; García-Álvarez, Begoña; Pérez-Gil, Jesús

2013-03-01

Pulmonary surfactant is a lipid-protein complex secreted by the respiratory epithelium of mammalian lungs, which plays an essential role in stabilising the alveolar surface and so reducing the work of breathing. The surfactant protein SP-B is part of this complex, and is strictly required for the assembly of pulmonary surfactant and its extracellular development to form stable surface-active films at the air-liquid alveolar interface, making the lack of SP-B incompatible with life. In spite of its physiological importance, a model for the structure and the mechanism of action of SP-B is still needed. The sequence of SP-B is homologous to that of the saposin-like family of proteins, which are membrane-interacting polypeptides with apparently diverging activities, from the co-lipase action of saposins to facilitate the degradation of sphingolipids in the lysosomes to the cytolytic actions of some antibiotic proteins, such as NK-lysin and granulysin or the amoebapore of Entamoeba histolytica. Numerous studies on the interactions of these proteins with membranes have still not explained how a similar sequence and a potentially related fold can sustain such apparently different activities. In the present review, we have summarised the most relevant features of the structure, lipid-protein and protein-protein interactions of SP-B and the saposin-like family of proteins, as a basis to propose an integrated model and a common mechanistic framework of the apparent functional versatility of the saposin fold.

University strategy for doctoral training: the Ghent University Doctoral Schools.

PubMed

Bracke, N; Moens, L

2010-01-01

The Doctoral Schools at Ghent University have a three-fold mission: (1) to provide support to doctoral students during their doctoral research, (2) to foster a quality culture in (doctoral) research, (3) to promote the international and social stature and prestige of the doctorate vis-a-vis potential researchers and the potential labour market. The Doctoral Schools offer top-level specialized courses and transferable skills training to doctoral students as part of their doctoral training programme. They establish mechanisms of quality assurance in doctoral research. The Doctoral Schools initialize and support initiatives of internationalization. They also organize information sessions, promotional events and interaction with the labour market, and as such keep a finger on the pulse of external stakeholders.

Bacillus subtilis affects miRNAs and flavanoids production in Agrobacterium-Tobacco interaction.

PubMed

Nazari, Fahimeh; Safaie, Naser; Soltani, Bahram Mohammad; Shams-Bakhsh, Masoud; Sharifi, Mohsen

2017-09-01

Agrobacterium tumefaciens is a very destructive plant pathogen. Selection of effective biological agents against this pathogen depends on more insight into molecular plant defence responses during the biocontrol agent-pathogen interaction. Auxin as a phytohormone is a key contributor in pathogenesis and plant defence and accumulation of auxin transport carriers are accompanied by increasing in flavonoid and miRNAs concentrations during plant interactions with bacteria. The aim of this research was molecular analysis of Bacillus subtilis (ATCC21332) biocontrol effect against A. tumefaciens (IBRC-M10701) pathogen interacting with Nicotiana tabacum plants. Tobacco plants were either treated with both or one of the challenging bacteria and the expression of miRNAs inside the plants were analysed through qRT-PCR. The results indicated that the bacterial treatments affect expression level of nta-miRNAs. In tobacco plants treated only with A. tumefaciens the expression of nta-miR393 was more than that was recorded for nta-miR167 (3.8 folds, P < 0.05 in 3dpi). While the expression level of nta-miR167 was more than the expression of nta-miR393 in other treatments including tobacco plants treated only with B. subtilis (2.1 folds, P < 0.05) and the plants treated with both of the bacteria (3.9 folds, P < 0.05) in 3 dpi. Also, the composition and concentration of rutin, myrecetin, daidzein and vitexin flavanoid derivatives were detected using HPLC and analysed according the standard curves. All of the tested flavanoid compounds were highly detected in Tobacco plants which were only challenged with A. tumefaciens. The amount of these compounds in the plants which were challenged with the B. subtilis alone, was similar to the amount recorded for the plants challenged with the both bacteria. This study suggests a relationship between the upregulation of nta-miR167, nta-miR393 and accumulation of flavanoid compounds. Overall, the expression of these miRNAs as well as flavonoid derivatives has the potential of being used as biomarkers for the interaction of B. subtilis and A. tumefaciens model system in N. tabacum. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Differences Between a Single- and a Double-Folding Nucleus-^{9}Be Optical Potential

NASA Astrophysics Data System (ADS)

Bonaccorso, A.; Carstoiu, F.; Charity, R. J.; Kumar, R.; Salvioni, G.

2016-05-01

We have recently constructed two very successful n-^9Be optical potentials (Bonaccorso and Charity in Phys Rev C89:024619, 2014). One by the Dispersive Optical Model (DOM) method and the other (AB) fully phenomenological. The two potentials have strong surface terms in common for both the real and the imaginary parts. This feature makes them particularly suitable to build a single-folded (light-) nucleus-^9Be optical potential by using ab-initio projectile densities such as those obtained with the VMC method (Wiringa http://www.phy.anl.gov/theory/research/density/). On the other hand, a VMC density together with experimental nucleon-nucleon cross-sections can be used also to obtain a neutron and/or proton-^9Be imaginary folding potential. We will use here an ab-initio VMC density (Wiringa http://www.phy.anl.gov/theory/research/density/) to obtain both a n-^9Be single-folded potential and a nucleus-nucleus double-folded potential. In this work we report on the cases of ^8B, ^8Li and ^8C projectiles. Our approach could be the basis for a systematic study of optical potentials for light exotic nuclei scattering on such light targets. Some of the projectiles studied are cores of other exotic nuclei for which neutron knockout has been used to extract spectroscopic information. For those cases, our study will serve to make a quantitative assessment of the core-target part of the reaction description, in particular its localization.

Accurately controlled sequential self-folding structures by polystyrene film

NASA Astrophysics Data System (ADS)

Deng, Dongping; Yang, Yang; Chen, Yong; Lan, Xing; Tice, Jesse

2017-08-01

Four-dimensional (4D) printing overcomes the traditional fabrication limitations by designing heterogeneous materials to enable the printed structures evolve over time (the fourth dimension) under external stimuli. Here, we present a simple 4D printing of self-folding structures that can be sequentially and accurately folded. When heated above their glass transition temperature pre-strained polystyrene films shrink along the XY plane. In our process silver ink traces printed on the film are used to provide heat stimuli by conducting current to trigger the self-folding behavior. The parameters affecting the folding process are studied and discussed. Sequential folding and accurately controlled folding angles are achieved by using printed ink traces and angle lock design. Theoretical analyses are done to guide the design of the folding processes. Programmable structures such as a lock and a three-dimensional antenna are achieved to test the feasibility and potential applications of this method. These self-folding structures change their shapes after fabrication under controlled stimuli (electric current) and have potential applications in the fields of electronics, consumer devices, and robotics. Our design and fabrication method provides an easy way by using silver ink printed on polystyrene films to 4D print self-folding structures for electrically induced sequential folding with angular control.

Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis

PubMed Central

Srivastava, Shikha; Somasagara, Ranganatha R.; Hegde, Mahesh; Nishana, Mayilaadumveettil; Tadi, Satish Kumar; Srivastava, Mrinal; Choudhary, Bibha; Raghavan, Sathees C.

2016-01-01

Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy. PMID:27068577

Assembly constraints drive co-evolution among ribosomal constituents.

PubMed

Mallik, Saurav; Akashi, Hiroshi; Kundu, Sudip

2015-06-23

Ribosome biogenesis, a central and essential cellular process, occurs through sequential association and mutual co-folding of protein-RNA constituents in a well-defined assembly pathway. Here, we construct a network of co-evolving nucleotide/amino acid residues within the ribosome and demonstrate that assembly constraints are strong predictors of co-evolutionary patterns. Predictors of co-evolution include a wide spectrum of structural reconstitution events, such as cooperativity phenomenon, protein-induced rRNA reconstitutions, molecular packing of different rRNA domains, protein-rRNA recognition, etc. A correlation between folding rate of small globular proteins and their topological features is known. We have introduced an analogous topological characteristic for co-evolutionary network of ribosome, which allows us to differentiate between rRNA regions subjected to rapid reconstitutions from those hindered by kinetic traps. Furthermore, co-evolutionary patterns provide a biological basis for deleterious mutation sites and further allow prediction of potential antibiotic targeting sites. Understanding assembly pathways of multicomponent macromolecules remains a key challenge in biophysics. Our study provides a 'proof of concept' that directly relates co-evolution to biophysical interactions during multicomponent assembly and suggests predictive power to identify candidates for critical functional interactions as well as for assembly-blocking antibiotic target sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Building toy models of proteins using coevolutionary information

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Raghunathan, Mohit; Onuchic, Jose

2015-03-01

Recent developments in global statistical methodologies have advanced the analysis of large collections of protein sequences for coevolutionary information. Coevolution between amino acids in a protein arises from compensatory mutations that are needed to maintain the stability or function of a protein over the course of evolution. This gives rise to quantifiable correlations between amino acid positions within the multiple sequence alignment of a protein family. Here, we use Direct Coupling Analysis (DCA) to infer a Potts model Hamiltonian governing the correlated mutations in a protein family to obtain the sequence-dependent interaction energies of a toy protein model. We demonstrate that this methodology predicts residue-residue interaction energies that are consistent with experimental mutational changes in protein stabilities as well as other computational methodologies. Furthermore, we demonstrate with several examples that DCA could be used to construct a structure-based model that quantitatively agrees with experimental data on folding mechanisms. This work serves as a potential framework for generating models of proteins that are enriched by evolutionary data that can potentially be used to engineer key functional motions and interactions in protein systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1427654).

Enthalpy-Driven RNA Folding: Single-Molecule Thermodynamics of Tetraloop–Receptor Tertiary Interaction†

PubMed Central

Fiore, Julie L.; Kraemer, Benedikt; Koberling, Felix; Edmann, Rainer; Nesbitt, David J.

2010-01-01

RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA tetraloop–receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21–47 °C), from which a van’t Hoff analysis yields the enthalpy (ΔH°) and entropy (ΔS°) of docking. Tetraloop–receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl2 and 100 mM NaCl, with excellent agreement between immobilized (ΔH° = −17.4 ± 1.6 kcal/mol, and ΔS° = −56.2 ± 5.4 cal mol−1 K−1) and freely diffusing (ΔH° = −17.2 ± 1.6 kcal/mol, and ΔS° = −55.9 ± 5.2 cal mol−1 K−1) species. Kinetic heterogeneity in the tetraloop–receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the tetraloop–receptor interaction can account for ~60% of the ΔH° and ΔS° of P4–P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated tetraloop–receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions. PMID:19186984

New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase.

PubMed

Oliveberg, M; Fersht, A R

1996-05-28

We use in this study a novel kinetic approach to determine the H+ titration properties of a semiburied salt link in the transition state for unfolding of barnase. The approach is based on changes in the pH dependence of the kinetics upon mutation of a target residue. This makes it relatively insensitive to the absolute value of the stability and, thereby, to artifacts caused by structural rearrangements around the site of mutation. The semiburied salt bridge studied here is between Asp93 and Arg69. Mutation of either residue significantly destabilized the protein, and the pKa value of Asp93 is severely lowered in the native state to below 1 because of the ionic interaction with Arg69. The Asp93-Arg69 salt link appears to be formed early in the folding process; the pKa value of Asp93 in the transition state (approximately 1) is similar to that in the native state, and deletion of the ionic interaction with Arg69 substantially destabilizes the folding intermediate and changes the kinetic behavior from multistate to two-state or close to two-state, depending on the conditions. The results suggest that the formation of ionic interactions within clusters of hydrophobic residues can be important for early folding events and can control kinetically the folding pathway. This is not because of the inherent stability of the salt link but because the presence of two unpaired charges is very unfavorable. The data reveal also that fractional phi values are consistent with a uniformly expanded transition state or one with closely spaced energy levels and not with parallel folding pathways.

Binding, folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing.

PubMed

Reid, Keon A; Davis, Caitlin M; Dyer, R Brian; Kindt, James T

2018-03-01

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKV d P l PTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a "flip and dip" mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploration of the folding dynamics of human telomeric G-quadruplex with a hybrid atomistic structure-based model

NASA Astrophysics Data System (ADS)

Bian, Yunqiang; Ren, Weitong; Song, Feng; Yu, Jiafeng; Wang, Jihua

2018-05-01

Structure-based models or Gō-like models, which are built from one or multiple particular experimental structures, have been successfully applied to the folding of proteins and RNAs. Recently, a variant termed the hybrid atomistic model advances the description of backbone and side chain interactions of traditional structure-based models, by borrowing the description of local interactions from classical force fields. In this study, we assessed the validity of this model in the folding problem of human telomeric DNA G-quadruplex, where local dihedral terms play important roles. A two-state model was developed and a set of molecular dynamics simulations was conducted to study the folding dynamics of sequence Htel24, which was experimentally validated to adopt two different (3 + 1) hybrid G-quadruplex topologies in K+ solution. Consistent with the experimental observations, the hybrid-1 conformation was found to be more stable and the hybrid-2 conformation was kinetically more favored. The simulations revealed that the hybrid-2 conformation folded in a higher cooperative manner, which may be the reason why it was kinetically more accessible. Moreover, by building a Markov state model, a two-quartet G-quadruplex state and a misfolded state were identified as competing states to complicate the folding process of Htel24. Besides, the simulations also showed that the transition between hybrid-1 and hybrid-2 conformations may proceed an ensemble of hairpin structures. The hybrid atomistic structure-based model reproduced the kinetic partitioning folding dynamics of Htel24 between two different folds, and thus can be used to study the complex folding processes of other G-quadruplex structures.

Role of Urea-Aromatic Stacking Interactions in Stabilizing the Aromatic Residues of the Protein in Urea-Induced Denatured State.

PubMed

Goyal, Siddharth; Chattopadhyay, Aditya; Kasavajhala, Koushik; Priyakumar, U Deva

2017-10-25

A delicate balance of different types of intramolecular interactions makes the folded states of proteins marginally more stable than the unfolded states. Experiments use thermal, chemical, or mechanical stress to perturb the folding equilibrium for examining protein stability and the protein folding process. Elucidation of the mechanism by which chemical denaturants unfold proteins is crucial; this study explores the nature of urea-aromatic interactions relevant in urea-assisted protein denaturation. Free energy profiles corresponding to the unfolding of Trp-cage miniprotein in the presence and absence of urea at three different temperatures demonstrate the distortion of the hydrophobic core to be a crucial step. Exposure of the Trp6 residue to the solvent is found to be favored in the presence of urea. Previous experiments showed that urea has a high affinity for aromatic groups of proteins. We show here that this is due to the remarkable ability of urea to form stacking and NH-π interactions with aromatic groups of proteins. Urea-nucleobase stacking interactions have been shown to be crucial in urea-assisted RNA unfolding. Examination of these interactions using microsecond-long unrestrained simulations shows that urea-aromatic stacking interactions are stabilizing and long lasting. Further MD simulations, thermodynamic integration, and quantum mechanical calculations on aromatic model systems reveal that such interactions are possible for all the aromatic amino acid side-chains. Finally, we validate the ubiquitous nature of urea-aromatic stacking interactions by analyzing experimental structures of urea transporters and proteins crystallized in the presence of urea or urea derivatives.

Evaluating the genotoxicity of topoisomerase-targeted antibiotics

PubMed Central

Smart, Daniel J.; Lynch, Anthony M.

2012-01-01

Antibiotics like fluoroquinolones (FQs) that target bacterial type II topoisomerases pose a potential genotoxic risk due to interactions with mammalian topoisomerase II (TOPO II) counterparts. Inhibition of TOPO II can lead to the generation of clastogenic DNA double-strand breaks (DSBs) that can in turn manifest in mutagenesis. Thus, methods that allow early identification of drugs that present the greatest hazard are warranted. A rapid, medium-throughput and predictive genotoxicity screen that can be applied to bacterial type II topoisomerase inhibitors is described herein. Maximal induction of the DSB biomarker serine139-phosphorylated histone H2AX (γH2AX) in L5178Y cells was quantified via flow cytometry and correlated with data derived from the mouse lymphoma screen (MLS), a default assay used to rank genotoxic potential. When applied to a class of novel bacterial type II topoisomerase inhibitors (NBTIs) in lead-optimisation, maximal γH2AX induction >1.4-fold (relative to controls) identified 22/27 NBTIs that induced >6-fold relative mutation frequency (MF) in MLS. Moreover, response signatures comprising of γH2AX induction and G2M cell cycle arrest elucidated using this approach suggested that these NBTIs, primarily of the H class, operated via a TOPO II poison-like mechanism of action (MoA) similar to FQs. NBTIs that induced ≤6-fold relative MF, which were mainly A class-derived, had less impact on γH2AX (≤1.4-fold) and also evoked G1 arrest, indicating that their cytotoxic effects were likely mediated through a non-poison MoA. Concordance between assays was 86% (54/63) when 1.4- and 6-fold ‘cut offs’ were applied. These findings were corroborated through inspection of human TOPO IIα IC50 data as NBTIs exhibiting equivalent inhibitory capacities had differing genotoxic potencies. Deployed in an early screening capacity, the γH2AX by flow assay coupled with structure–activity relationship evaluation can provide insight into MoA and impact medicinal chemistry efforts, ultimately leading to the production of inherently safer molecules. PMID:22155972

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

PubMed

Robinson, Philip J; Pringle, Marie Anne; Woolhead, Cheryl A; Bulleid, Neil J

2017-04-28

The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Dominant Folding Route Minimizes Backbone Distortion in SH3

PubMed Central

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

2012-01-01

Energetic frustration in protein folding is minimized by evolution to create a smooth and robust energy landscape. As a result the geometry of the native structure provides key constraints that shape protein folding mechanisms. Chain connectivity in particular has been identified as an essential component for realistic behavior of protein folding models. We study the quantitative balance of energetic and geometrical influences on the folding of SH3 in a structure-based model with minimal energetic frustration. A decomposition of the two-dimensional free energy landscape for the folding reaction into relevant energy and entropy contributions reveals that the entropy of the chain is not responsible for the folding mechanism. Instead the preferred folding route through the transition state arises from a cooperative energetic effect. Off-pathway structures are penalized by excess distortion in local backbone configurations and contact pair distances. This energy cost is a new ingredient in the malleable balance of interactions that controls the choice of routes during protein folding. PMID:23166485

Structural Basis of the Induced-Fit Mechanism of 1,4-Dihydroxy-2-Naphthoyl Coenzyme A Synthase from the Crotonase Fold Superfamily

PubMed Central

Li, Jie; Li, Yan; Jiang, Ming; Zhou, Jiahai; Guo, Zhihong

2013-01-01

1, 4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase fold enzyme with an implicated role of conformational changes in catalysis. We have identified these conformational changes by determining the structures of its Escherichia coli and Synechocystis sp. PCC6803 orthologues in complex with a product analog. The structural changes include the folding of an active-site loop into a β-hairpin and significant reorientation of a helix at the carboxy terminus. Interestingly, a new interface is formed between the ordered loop and the reoriented helix, both of which also form additional interactions with the coenzyme A moiety of the ligand. Site-directed mutation of the amino acid residues involved in these ligand-induced interactions significantly diminishes the enzyme activity. These results suggest a catalytically essential induced-fit that is likely initiated by the enzyme-ligand interactions at the active site. PMID:23658663

A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

PubMed Central

Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

2014-01-01

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

Anomalous Diffusion Reports on the Interaction of Misfolded Proteins with the Quality Control Machinery in the Endoplasmic Reticulum

PubMed Central

Malchus, Nina; Weiss, Matthias

2010-01-01

A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER. PMID:20713018

An extracellular disulfide bond forming protein (DsbF) from Mycobacterium tuberculosis: Structural, biochemical and gene expression analysis

PubMed Central

Chim, Nicholas; Riley, Robert; The, Juliana; Im, Soyeon; Segelke, Brent; Lekin, Tim; Yu, Minmin; Hung, Li Wei; Terwilliger, Tom; Whitelegge, Julian P.; Goulding, Celia W.

2010-01-01

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here we present structural, biochemical and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pKa for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein co-expression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicate anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions. PMID:20060836

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

PubMed Central

Phillips, J J; Javadi, Y; Millership, C; Main, E R G

2012-01-01

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium. PMID:22170589

The Energy Landscape, Folding Pathways and the Kinetics of a Knotted Protein

PubMed Central

Prentiss, Michael C.; Wales, David J.; Wolynes, Peter G.

2010-01-01

The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly. PMID:20617197

High diversification of CD94 by alternative splicing in New World primates.

PubMed

Galindo, John A; Cadavid, Luis F

2013-04-01

CD94 forms heterodimers with NKG2A, -C, or -E to constitute lectin-like natural killer cell receptors for MHC-E. Its structure differs from other C-type lectins in that the second α-helix is replaced by a loop that forms the interacting interface with the NKG2 molecules. Although CD94 has remained highly conserved mammals, several alternative splicing variants have been detected in some species. To evaluate the prevalence and significance of this phenomenon, we have cloned and sequenced CD94 cDNAs in six species of New World primates from the Cebidae and Atelidae families. Full-length sequences had a mean similarity of 96 % amongst New World primates and of 90 % to the human orthologue, with little variation in the residues interacting with NKG2 or MHC-E molecules. Despite this high conservation, a total of 14 different splice variants were identified, half of which were shared by two or more primate species. Homology-based modeling of the C-type lectin domain showed that most isoforms folded stably, although they had modifications that prevented its interaction with NKG2 and MHC-E. Two isoforms were predicted to replace the typical CD94 loop by a second α-helix, evidencing a domain fold transition from a CD94 structure to a canonical C-type lectin. These two structures were more similar to members of the CLEC lectin family than to the native CD94. Thus, CD94 has remained conserved in primates to maintain functional interactions with NKG2 and MHC-E, while at the same time has diversified by alternative splicing potentially providing additional functional scenarios.

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

PubMed Central

BRADRICK, THOMAS D.; MARINO, JOHN P.

2004-01-01

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (Δ TAR-ap23 and Δ TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP ΔTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD ~ 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD ~ 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the ΔTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD ~ 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD ~ 4 μM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions. PMID:15273324

Interaction of β-Sheet Folds with a Gold Surface

PubMed Central

Hoefling, Martin; Monti, Susanna; Corni, Stefano; Gottschalk, Kay Eberhard

2011-01-01

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance. PMID:21687744

Corrector VX-809 promotes interactions between cytoplasmic loop one and the first nucleotide-binding domain of CFTR.

PubMed

Loo, Tip W; Clarke, David M

2017-07-15

A large number of correctors have been identified that can partially repair defects in folding, stability and trafficking of CFTR processing mutants that cause cystic fibrosis (CF). The best corrector, VX-809 (Lumacaftor), has shown some promise when used in combination with a potentiator (Ivacaftor). Understanding the mechanism of VX-809 is essential for development of better correctors. Here, we tested our prediction that VX-809 repairs folding and processing defects of CFTR by promoting interactions between the first cytoplasmic loop (CL1) of transmembrane domain 1 (TMD1) and the first nucleotide-binding domain (NBD1). To investigate whether VX-809 promoted CL1/NBD1 interactions, we performed cysteine mutagenesis and disulfide cross-linking analysis of Cys-less TMD1 (residues 1-436) and ΔTMD1 (residues 437-1480; NBD1-R-TMD2-NBD2) truncation mutants. It was found that VX-809, but not bithiazole correctors, promoted maturation (exited endoplasmic reticulum for addition of complex carbohydrate in the Golgi) of the ΔTMD1 truncation mutant only when it was co-expressed in the presence of TMD1. Expression in the presence of VX-809 also promoted cross-linking between R170C (in CL1 of TMD1 protein) and L475C (in NBD1 of the ΔTMD1 truncation protein). Expression of the ΔTMD1 truncation mutant in the presence of TMD1 and VX-809 also increased the half-life of the mature protein in cells. The results suggest that the mechanism by which VX-809 promotes maturation and stability of CFTR is by promoting CL1/NBD1 interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Electronic polarization stabilizes tertiary structure prediction of HP-36.

PubMed

Duan, Li L; Zhu, Tong; Zhang, Qing G; Tang, Bo; Zhang, John Z H

2014-04-01

Molecular dynamic (MD) simulations with both implicit and explicit solvent models have been carried out to study the folding dynamics of HP-36 protein. Starting from the extended conformation, the secondary structure of all three helices in HP-36 was formed in about 50 ns and remained stable in the remaining simulation. However, the formation of the tertiary structure was difficult. Although some intermediates were close to the native structure, the overall conformation was not stable. Further analysis revealed that the large structure fluctuation of loop and hydrophobic core regions was devoted mostly to the instability of the structure during MD simulation. The backbone root-mean-square deviation (RMSD) of the loop and hydrophobic core regions showed strong correlation with the backbone RMSD of the whole protein. The free energy landscape indicated that the distribution of main chain torsions in loop and turn regions was far away from the native state. Starting from an intermediate structure extracted from the initial AMBER simulation, HP-36 was found to generally fold to the native state under the dynamically adjusted polarized protein-specific charge (DPPC) simulation, while the peptide did not fold into the native structure when AMBER force filed was used. The two best folded structures were extracted and taken into further simulations in water employing AMBER03 charge and DPPC for 25 ns. Result showed that introducing polarization effect into interacting potential could stabilize the near-native protein structure.

Patient-Specific Computational Modeling of Human Phonation

NASA Astrophysics Data System (ADS)

Xue, Qian; Zheng, Xudong; University of Maine Team

2013-11-01

Phonation is a common biological process resulted from the complex nonlinear coupling between glottal aerodynamics and vocal fold vibrations. In the past, the simplified symmetric straight geometric models were commonly employed for experimental and computational studies. The shape of larynx lumen and vocal folds are highly three-dimensional indeed and the complex realistic geometry produces profound impacts on both glottal flow and vocal fold vibrations. To elucidate the effect of geometric complexity on voice production and improve the fundamental understanding of human phonation, a full flow-structure interaction simulation is carried out on a patient-specific larynx model. To the best of our knowledge, this is the first patient-specific flow-structure interaction study of human phonation. The simulation results are well compared to the established human data. The effects of realistic geometry on glottal flow and vocal fold dynamics are investigated. It is found that both glottal flow and vocal fold dynamics present a high level of difference from the previous simplified model. This study also paved the important step toward the development of computer model for voice disease diagnosis and surgical planning. The project described was supported by Grant Number ROlDC007125 from the National Institute on Deafness and Other Communication Disorders (NIDCD).

A new model for approximating RNA folding trajectories and population kinetics

NASA Astrophysics Data System (ADS)

Kirkpatrick, Bonnie; Hajiaghayi, Monir; Condon, Anne

2013-01-01

RNA participates both in functional aspects of the cell and in gene regulation. The interactions of these molecules are mediated by their secondary structure which can be viewed as a planar circle graph with arcs for all the chemical bonds between pairs of bases in the RNA sequence. The problem of predicting RNA secondary structure, specifically the chemically most probable structure, has many useful and efficient algorithms. This leaves RNA folding, the problem of predicting the dynamic behavior of RNA structure over time, as the main open problem. RNA folding is important for functional understanding because some RNA molecules change secondary structure in response to interactions with the environment. The full RNA folding model on at most O(3n) secondary structures is the gold standard. We present a new subset approximation model for the full model, give methods to analyze its accuracy and discuss the relative merits of our model as compared with a pre-existing subset approximation. The main advantage of our model is that it generates Monte Carlo folding pathways with the same probabilities with which they are generated under the full model. The pre-existing subset approximation does not have this property.

Revealing the distinct folding phases of an RNA three-helix junction.

PubMed

Plumridge, Alex; Katz, Andrea M; Calvey, George D; Elber, Ron; Kirmizialtin, Serdal; Pollack, Lois

2018-05-14

Remarkable new insight has emerged into the biological role of RNA in cells. RNA folding and dynamics enable many of these newly discovered functions, calling for an understanding of RNA self-assembly and conformational dynamics. Because RNAs pass through multiple structures as they fold, an ensemble perspective is required to visualize the flow through fleetingly populated sets of states. Here, we combine microfluidic mixing technology and small angle X-ray scattering (SAXS) to measure the Mg-induced folding of a small RNA domain, the tP5abc three helix junction. Our measurements are interpreted using ensemble optimization to select atomically detailed structures that recapitulate each experimental curve. Structural ensembles, derived at key stages in both time-resolved studies and equilibrium titrations, reproduce the features of known intermediates, and more importantly, offer a powerful new structural perspective on the time-progression of folding. Distinct collapse phases along the pathway appear to be orchestrated by specific interactions with Mg ions. These key interactions subsequently direct motions of the backbone that position the partners of tertiary contacts for later bonding, and demonstrate a remarkable synergy between Mg and RNA across numerous time-scales.

An ABI3-interactor of conifers responds to multiple hormones.

PubMed

Zeng, Ying; Zhao, Tiehan; Kermode, Allison

2013-11-01

CnAIP2 (Callitropsis nootkatensis ABI3-Interacting Protein 2) was previously identified as a protein that interacts with the yellow-cedar ABI3 protein. CnAIP2 plays important roles during several key transitions of the plant lifecycle and acts as a global regulator with functions opposite to those of ABI3 proteins. Here we report that the CnAIP2 gene promoter is strongly upregulated by all of the major plant hormones. Young Arabidopsis seedlings expressing a chimeric CnAIP2pro-GUS construct were subjected to exogenously applied hormones; the maximum fold-enhancement of GUS activity was as high as 47-fold, and each hormone showed a distinctive cell/tissue-specific pattern of GUS induction. By far the greatest response was elicited by the synthetic auxin 2,4-D (47-fold induction); the other hormones tested stimulated GUS activities by 8- to 21-fold. The CnAIP2 promoter also responded to glucose and salt (NaCl), albeit to a lesser extent (2- to 3-fold induction). As well as acting in an antagonistic way to the global regulator ABI3, CnAIP2 appears to participate in multiple hormonal crosstalk pathways to carry out its functions.

When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches

PubMed Central

Muñoz, Victor; Cerminara, Michele

2016-01-01

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. PMID:27574021

Nature of Driving Force for Protein Folding: A Result From Analyzing the Statistical Potential

NASA Astrophysics Data System (ADS)

Li, Hao; Tang, Chao; Wingreen, Ned S.

1997-07-01

In a statistical approach to protein structure analysis, Miyazawa and Jernigan derived a 20×20 matrix of inter-residue contact energies between different types of amino acids. Using the method of eigenvalue decomposition, we find that the Miyazawa-Jernigan matrix can be accurately reconstructed from its first two principal component vectors as Mij = C0+C1\\(qi+qj\\)+C2qiqj, with constant C's, and 20 q values associated with the 20 amino acids. This regularity is due to hydrophobic interactions and a force of demixing, the latter obeying Hildebrand's solubility theory of simple liquids.

Chemopreventive potential of in vitro fermented nuts in LT97 colon adenoma and primary epithelial colon cells.

PubMed

Schlörmann, Wiebke; Lamberty, Julia; Lorkowski, Stefan; Ludwig, Diana; Mothes, Henning; Saupe, Christian; Glei, Michael

2017-05-01

Due to their beneficial nutritional profile the consumption of nuts contributes to a healthy diet and might reduce colon cancer risk. To get closer insights into potential mechanisms, the chemopreventive potential of different in vitro fermented nut varieties regarding the modulation of genes involved in detoxification (CAT, SOD2, GSTP1, GPx1) and cell cycle (p21, cyclin D2) as well as proliferation and apoptosis was examined in LT97 colon adenoma and primary epithelial colon cells. Fermentation supernatants (FS) of nuts significantly induced mRNA expression of CAT (up to 4.0-fold), SOD2 (up to 2.5-fold), and GSTP1 (up to 2.3-fold), while GPx1 expression was significantly reduced by all nut FS (0.8 fold on average). Levels of p21 mRNA were significantly enhanced (up to 2.6-fold), whereas all nut FS significantly decreased cyclin D2 expression (0.4-fold on average). In primary epithelial cells, expression of CAT (up to 3.5-fold), GSTP1 (up to 3.0-fold), and GPx1 (up to 3.9-fold) was increased, whereas p21 and cyclin D2 levels were not influenced. Nut FS significantly inhibited growth of LT97 cells and increased levels of early apoptotic cells (8.4% on average) and caspase 3 activity (4.6-fold on average), whereas caspase 3 activity was not modulated in primary colon cells. The differential modulation of genes involved in detoxification and cell cycle together with an inhibition of proliferation and induction of apoptosis in adenoma cells might contribute to chemopreventive effects of nuts regarding colon cancer. © 2017 Wiley Periodicals, Inc.

Semimicroscopic analysis of 6Li+28Si elastic scattering at 76 to 318 MeV

NASA Astrophysics Data System (ADS)

Hassanain, M. A.; Anwar, M.; Behairy, Kassem O.

2018-04-01

Using the α-cluster structure of colliding nuclei, the elastic scattering of 6Li+28Si at energies from 76 to 318 MeV has been investigated by the use of the real folding cluster approach. The results of the cluster analysis are compared with those obtained by the CDM3Y6 effective density- and energy-dependent nucleon-nucleon (NN) interaction based upon G -matrix elements of the M3Y-Paris potential. A Woods-Saxon (WS) form was used for the imaginary potential. For all energies and derived potentials, the diffraction region was well reproduced, except at Elab=135 and 154 MeV at large angle. These results suggest that the addition of the surface (DWS) imaginary potential term to the volume imaginary potential is essential for a correct description of the refractive structure of the 6Li elastic scattering distribution at these energies. The energy dependence of the total reaction cross sections and that of the real and imaginary volume integrals is also discussed.

Ex Vivo and in Vivo Evaluation of the Effect of Coating a Coumarin-6-Labeled Nanostructured Lipid Carrier with Chitosan-N-acetylcysteine on Rabbit Ocular Distribution.

PubMed

Liu, Dandan; Li, Jinyu; Cheng, Bingchao; Wu, Qingyin; Pan, Hao

2017-08-07

This study is focused on further understanding the characteristics of chitosan-N-acetylcysteine surface-modified nanostructured lipid carriers (CS-NAC-NLCs) in their interaction with ocular mucosa. Coumarin-6 (C6)-labeled NLCs, including uncoated NLCs, chitosan hydrochloride (CH)-, and CS-NAC-coated NLCs, were developed using a melt-emulsification technique and subsequently decorated with different types or portions of chitosan derivatives. Mucoadhesion was evaluated ex vivo using a flow-through process with fluorescence detection. The results demonstrated that the presence of CS-NAC on the C6-NLC surface provided the most obvious enhancement in adhesion due to the formation of both noncovalent (ionic) and covalent (disulfide bridges) interactions with mucus chains. Meanwhile, the concentration of CS-NAC in the formulation positively influenced the viscosity of the nanoparticles and hence prolonged their retention in the ocular tissue. Transcorneal penetration studies revealed that CS-NAC-NLC particles were able to penetrate through the entire corneal epithelium primarily via a transcellular route. The transport depth and velocity strongly relied on the modification material and the particle size. Ex vivo fluorescence imaging and in vivo ocular distribution investigations showed that C6 was broadly distributed in rabbit eye tissues and absorbed by aqueous humor after CS-NAC-NLC instillation. In relation to C6 eye drops, CS-NAC-NLCs achieved considerably higher C max (4.01-fold), MRT 0-∞ (1.87-fold), and AUC 0-∞ (16.29-fold) in the aqueous humor. Moreover, the increase in drug absorption was greater in the cornea than in the conjunctiva. Thereby, it is possible to draw a conclusion that CS-NAC-NLCs presented great potential for drug application to the front portion of the eye.

Rational and Modular Design of Potent Ligands Targeting the RNA that Causes Myotonic Dystrophy 2

PubMed Central

Lee, Melissa M.; Pushechnikov, Alexei; Disney, Matthew D.

2009-01-01

Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6′-N-5-hexynoate kanamycin A (1) prefers to bind 2×2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops were found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC50 of 25 nM. This ligand is higher affinity and more specific for binding DM2 RNA than MBNL-1. It binds the DM2 RNA at least 20-times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6′-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNA-protein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable. PMID:19348464

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

PubMed

Bähring, R; Dannenberg, J; Peters, H C; Leicher, T; Pongs, O; Isbrandt, D

2001-06-29

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Interactions of tillage and cover crop on water, sediment, and pre-emergence herbicide loss in glyphosate-resistant cotton: implications for the control of glyphosate-resistant weed biotypes.

PubMed

Krutz, L Jason; Locke, Martin A; Steinriede, R Wade

2009-01-01

The need to control glyphosate [N-(phosphonomethyl)glycine]-resistant weed biotypes with tillage and preemergence herbicides in glyphosate-resistant crops (GRCs) is causing a reduction in no-tillage hectarage thereby threatening the advances made in water quality over the past decade. Consequently, if environmental gains afforded by GRCs are to be maintained, then an in-field best management practice (BMP) compatible with tillage is required for hectarage infested with glyphosate-resistant weed biotypes. Thus, 1 d after a preemergent application of fluometuron [N,N-dimethyl-N'-(3-(trifluoromethyl)phenyl)urea] (1.02 kg ha(-1)) and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] (1.18 kg ha(-1)) to a Dundee silt loam (fine-silty, mixed, active, thermic Typic Endoaqualf), simulated rainfall (60 mm h(-1)) was applied to 0.0002-ha microplots for approximately 1.25 h to elucidate tillage (no tillage [NT] and reduced tillage [RT])and cover crop (no cover [NC] and rye cover [RC]) effects on water, sediment, and herbicide loss in surface runoff. Regardless of tillage, RC delayed time-to-runoff 1.3-fold, reduced cumulative runoff volume 1.4-fold, and decreased cumulative sediment loss 4.7-fold. Cumulative fluometuron loss was not affected by tillage or cover crop. Conversely, total metolachlor loss was 1.3-fold lower in NT than RT and 1.4-fold lower in RC than NC. These data indicate that RC can be established in hectarage requiring tillage and potentially curtail water, sediment, and preemergence herbicide losses in the spring to levels equivalent to or better than that of NT, thereby protecting environmental gains provided by GRCs.

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration*

PubMed Central

Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M.; Verma, Vikas K.; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C.; Shah, Vijay H.

2015-01-01

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. PMID:26534962

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

PubMed

Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

2015-12-25

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Altered vocal fold kinematics in synthetic self-oscillating models that employ adipose tissue as a lateral boundary condition.

NASA Astrophysics Data System (ADS)

Saidi, Hiba; Erath, Byron D.

2015-11-01

The vocal folds play a major role in human communication by initiating voiced sound production. During voiced speech, the vocal folds are set into sustained vibrations. Synthetic self-oscillating vocal fold models are regularly employed to gain insight into flow-structure interactions governing the phonation process. Commonly, a fixed boundary condition is applied to the lateral, anterior, and posterior sides of the synthetic vocal fold models. However, physiological observations reveal the presence of adipose tissue on the lateral surface between the thyroid cartilage and the vocal folds. The goal of this study is to investigate the influence of including this substrate layer of adipose tissue on the dynamics of phonation. For a more realistic representation of the human vocal folds, synthetic multi-layer vocal fold models have been fabricated and tested while including a soft lateral layer representative of adipose tissue. Phonation parameters have been collected and are compared to those of the standard vocal fold models. Results show that vocal fold kinematics are affected by adding the adipose tissue layer as a new boundary condition.

On the Origin of Protein Superfamilies and Superfolds

NASA Astrophysics Data System (ADS)

Magner, Abram; Szpankowski, Wojciech; Kihara, Daisuke

2015-02-01

Distributions of protein families and folds in genomes are highly skewed, having a small number of prevalent superfamiles/superfolds and a large number of families/folds of a small size. Why are the distributions of protein families and folds skewed? Why are there only a limited number of protein families? Here, we employ an information theoretic approach to investigate the protein sequence-structure relationship that leads to the skewed distributions. We consider that protein sequences and folds constitute an information theoretic channel and computed the most efficient distribution of sequences that code all protein folds. The identified distributions of sequences and folds are found to follow a power law, consistent with those observed for proteins in nature. Importantly, the skewed distributions of sequences and folds are suggested to have different origins: the skewed distribution of sequences is due to evolutionary pressure to achieve efficient coding of necessary folds, whereas that of folds is based on the thermodynamic stability of folds. The current study provides a new information theoretic framework for proteins that could be widely applied for understanding protein sequences, structures, functions, and interactions.

Competition between surface adsorption and folding of fibril-forming polypeptides

NASA Astrophysics Data System (ADS)

Ni, Ran; Kleijn, J. Mieke; Abeln, Sanne; Cohen Stuart, Martien A.; Bolhuis, Peter G.

2015-02-01

Self-assembly of polypeptides into fibrillar structures can be initiated by planar surfaces that interact favorably with certain residues. Using a coarse-grained model, we systematically studied the folding and adsorption behavior of a β -roll forming polypeptide. We find that there are two different folding pathways depending on the temperature: (i) at low temperature, the polypeptide folds in solution into a β -roll before adsorbing onto the attractive surface; (ii) at higher temperature, the polypeptide first adsorbs in a disordered state and folds while on the surface. The folding temperature increases with increasing attraction as the folded β -roll is stabilized by the surface. Surprisingly, further increasing the attraction lowers the folding temperature again, as strong attraction also stabilizes the adsorbed disordered state, which competes with folding of the polypeptide. Our results suggest that to enhance the folding, one should use a weakly attractive surface. They also explain the recent experimental observation of the nonmonotonic effect of charge on the fibril formation on an oppositely charged surface [C. Charbonneau et al., ACS Nano 8, 2328 (2014), 10.1021/nn405799t].

The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods.

PubMed

Yu, Hongbo; Guo, Guoning; Zhang, Xiaoyu; Yan, Keliang; Xu, Chunyan

2009-11-01

Selective white-rot fungi have shown potential for lignocellulose pretreatment. In the study, a new fungal isolate, Echinodontium taxodii 2538, was used in biological pretreatment to enhance the enzymatic hydrolysis of two native woods: Chinese willow (hardwood) and China-fir (softwood). E. taxodii preferentially degraded the lignin during the pretreatment, and the pretreated woods showed significant increases in enzymatic hydrolysis ratios (4.7-fold for hardwood and 6.3-fold for softwood). To better understand effects of biological pretreatment on enzymatic hydrolysis, enzyme-substrate interactions were investigated. It was observed that E. taxodii enhanced initial adsorption of cellulase but which did not always translate to high initial hydrolysis rate. However, the rate of change in hydrolysis rate declined dramatically with decreasing irreversible adsorption of cellulase. Thus, the enhancement of enzymatic hydrolysis was attributed to the decline of irreversible adsorption which may result from partial lignin degradation and alteration in lignin structure after biological pretreatment.

Yeast mannoproteins improve thermal stability of anthocyanins at pH 7.0.

PubMed

Wu, Jine; Guan, Yongguang; Zhong, Qixin

2015-04-01

Anthocyanins are food colourants with strong antioxidant activities, but poor thermal stability limits their application in neutral foods. In the present study, impacts of yeast mannoproteins on the thermal stability of anthocyanins were studied at pH 7.0. The degradation of anthocyanins at 80 and 126 °C followed first order kinetics, and the addition of mannoproteins reduced the degradation rate constant and increased the half-life by 4 to 5-fold. After heating at 80 and 126 °C for 30 min, mannoproteins improved the colour stability of anthocyanins by 4 to 5-fold and maintained the antioxidant capacity of anthocyanins. Visible light absorption, fluorescence spectroscopy, and zeta-potential results suggest that anthocyanins bound with the protein moiety of mannoproteins by hydrophobic interactions, and that the inclusion of anthocyanins in complexes effectively reduced the thermal degradation at pH 7.0. Therefore, mannoproteins may expand the application of anthocyanins as natural colours or functional ingredients. Copyright © 2014 Elsevier Ltd. All rights reserved.

Robust Denaturation of Villin Headpiece by MoS2 Nanosheet: Potential Molecular Origin of the Nanotoxicity

NASA Astrophysics Data System (ADS)

Gu, Zonglin; Yang, Zaixing; Kang, Seung-Gu; Yang, Jerry R.; Luo, Judong; Zhou, Ruhong

2016-06-01

MoS2 nanosheet, a new two-dimensional transition metal dichalcogenides nanomaterial, has attracted significant attentions lately due to many potential promising biomedical applications. Meanwhile, there is also a growing concern on its biocompatibility, with little known on its interactions with various biomolecules such as proteins. In this study, we use all-atom molecular dynamics simulations to investigate the interaction of a MoS2 nanosheet with Villin Headpiece (HP35), a model protein widely used in protein folding studies. We find that MoS2 exhibits robust denaturing capability to HP35, with its secondary structures severely destroyed within hundreds of nanosecond simulations. Both aromatic and basic residues are critical for the protein anchoring onto MoS2 surface, which then triggers the successive protein unfolding process. The main driving force behind the adsorption process is the dispersion interaction between protein and MoS2 monolayer. Moreover, water molecules at the interface between some key hydrophobic residues (e.g. Trp-64) and MoS2 surface also help to accelerate the process driven by nanoscale drying, which provides a strong hydrophobic force. These findings might have shed new light on the potential nanotoxicity of MoS2 to proteins with atomic details, which should be helpful in guiding future biomedical applications of MoS2 with its nanotoxicity mitigated.

Identification and structural mechanism for a novel interaction between a ubiquitin ligase WWP1 and Nogo-A, a key inhibitor for central nervous system regeneration.

PubMed

Qin, Haina; Pu, Helen X; Li, Minfen; Ahmed, Sohail; Song, Jianxing

2008-12-23

Nogo-A has been extensively demonstrated to play key roles in inhibiting central nervous system regeneration, regulating endoplasmic reticulum formation, and maintaining the integrity of the neuromuscular junction. In this study, an E3 ubiquitin ligase WWP1 was first identified to be a novel interacting partner for Nogo-A both in vitro and in vivo. By using CD, ITC, and NMR, we have further conducted extensive studies on all four WWP1 WW domains and their interactions with a Nogo-A peptide carrying the only PPxY motif. The results lead to several striking findings. (1) Despite containing an unstructured region, the 186-residue WWP1 fragment containing all four WW domains is able to interact with the Nogo-A(650-666) peptide with a high affinity, with a dissociation constant (K(d)) of 1.68 microM. (2) Interestingly, four isolated WW domains show differential structural properties in the free states. WW1 and WW2 are only partially folded, while WW4 is well-folded. Nevertheless, they all become well-folded upon binding to Nogo-A(650-666), with K(d) values ranging from 1.03 to 3.85 microM. (3) The solution structure of the best-folded WW4 domain is determined, and the binding-perturbed residues were derived for both WW4 and Nogo-A(650-666) by NMR HSQC titrations. Moreover, on the basis of the NMR data, the complex model is constructed by HADDOCK 2.0. This study provides rationales as well as a template Nogo-A(650-666) for further design of molecules to intervene in the WWP1-Nogo-A interaction which may regulate the Nogo-A protein level by controlling its ubiquitination.

Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum.

PubMed Central

Di Jeso, Bruno; Ulianich, Luca; Pacifico, Francesco; Leonardi, Antonio; Vito, Pasquale; Consiglio, Eduardo; Formisano, Silvestro; Arvan, Peter

2003-01-01

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca(2+) from the ER elicited by thapsigargin (a specific inhibitor of ER Ca(2+)-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca(2+) strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER. PMID:12401114

One Peptide Reveals the Two Faces of α-Helix Unfolding-Folding Dynamics.

PubMed

Jesus, Catarina S H; Cruz, Pedro F; Arnaut, Luis G; Brito, Rui M M; Serpa, Carlos

2018-04-12

The understanding of fast folding dynamics of single α-helices comes mostly from studies on rationally designed peptides displaying sequences with high helical propensity. The folding/unfolding dynamics and energetics of α-helix conformations in naturally occurring peptides remains largely unexplored. Here we report the study of a protein fragment analogue of the C-peptide from bovine pancreatic ribonuclease-A, RN80, a 13-amino acid residue peptide that adopts a highly populated helical conformation in aqueous solution. 1 H NMR and CD structural studies of RN80 showed that α-helix formation displays a pH-dependent bell-shaped curve, with a maximum near pH 5, and a large decrease in helical content in alkaline pH. The main forces stabilizing this short α-helix were identified as a salt bridge formed between Glu-2 and Arg-10 and the cation-π interaction involving Tyr-8 and His-12. Thus, deprotonation of Glu-2 or protonation of His-12 are essential for the RN80 α-helix stability. In the present study, RN80 folding and unfolding were triggered by laser-induced pH jumps and detected by time-resolved photoacoustic calorimetry (PAC). The photoacid proton release, amino acid residue protonation, and unfolding/folding events occur at different time scales and were clearly distinguished using time-resolved PAC. The partial unfolding of the RN80 α-helix, due to protonation of Glu-2 and consequent breaking of the stabilizing salt bridge between Glu-2 and Arg-10, is characterized by a concentration-independent volume expansion in the sub-microsecond time range (0.8 mL mol -1 , 369 ns). This small volume expansion reports the cost of peptide backbone rehydration upon disruption of a solvent-exposed salt bridge, as well as backbone intrinsic expansion. On the other hand, RN80 α-helix folding triggered by His-12 protonation and subsequent formation of a cation-π interaction leads to a microsecond volume contraction (-6.0 mL mol -1 , ∼1.7 μs). The essential role of two discrete side chain interactions, a salt bridge, and in particular a single cation-π interaction in the folding dynamics of a naturally occurring α-helix peptide is uniquely revealed by these data.

Effect of the geometry of confining media on the stability and folding rate of α -helix proteins

NASA Astrophysics Data System (ADS)

Wang, Congyue; Piroozan, Nariman; Javidpour, Leili; Sahimi, Muhammad

2018-05-01

Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding temperature Tf on the size D of a confined medium occurs in larger media for larger proteins is correct only in spherical geometry, whereas the opposite is true in the two other geometries that we study. Also studied is the effect of the strength of the interaction between the confined media's walls and the proteins. If the walls are only weakly or moderately attractive, a complex behavior emerges that depends on the size of the confining medium.

Modeling of wind gap formation and development of sedimentary basins during fold growth: application to the Zagros Fold Belt, Iran.

NASA Astrophysics Data System (ADS)

Collignon, Marine; Yamato, Philippe; Castelltort, Sébastien; Kaus, Boris

2016-04-01

Mountain building and landscape evolution are controlled by the interactions between river dynamics and tectonic forces. Such interactions have been largely studied but a quantitative evaluation of tectonic/geomorphic feedbacks remains required for understanding sediments routing within orogens and fold-and-thrust belts. Here, we employ numerical simulations to assess the conditions of uplift and river incision necessary to deflect an antecedent drainage network during the growth of one or several folds. We propose that a partitioning of the river network into internal (endorheic) and longitudinal drainage arises as a result of lithological differences within the deforming crustal sedimentary cover. We show with examples from the Zagros Fold Belt (ZFB) that drainage patterns can be linked to the incision ratio R between successive lithological layers, corresponding to the ratio between their relative erodibilities or incision coefficients. Transverse drainage networks develop for uplift rates smaller than 0.8 mm.yr-1 and -10 < R < 10. Intermediate drainage network are obtained for uplift rates up to 2 mm.yr-1 and incision ratios of 20. Parallel drainage networks and formation of sedimentary basins occur for large values of incision ratio (R >20) and uplift rates between 1 and 2 mm.yr-1. These results have implications for predicting the distribution of sediment depocenters in fold-and-thrust belts, which can be of direct economic interest for hydrocarbon exploration.

Flow-structure interaction simulation of voice production in a canine larynx

NASA Astrophysics Data System (ADS)

Jiang, Weili; Zheng, Xudong; Xue, Qian; Oren, Liran; Khosla, Sid

2017-11-01

Experimental measurements conducted on a hemi-larynx canine vocal fold showed that negative pressures formed in the glottis near the superior surface of the vocal fold in the closing phase even without a supra-glottal vocal tract. It was hypothesized that such negative pressures were due to intraglottal vortices caused by flow separation in a divergent vocal tract during vocal fold closing phase. This work aims to test this hypothesis from the numerical aspect. Flow-structure interaction simulations are performed in realistic canine laryngeal shapes. In the simulations, a sharp interface immersed boundary method based incompressible flow solver is utilized to model the air flow; a finite element based solid mechanics solver is utilized to model the vocal fold vibration. The geometric structure of the vocal fold and vocal tract are based on MRI scans of a mongrel canine. The vocal fold tissue is modeled as transversely isotropic nonlinear materials with a vertical stiffness gradient. Numerical indentation is first performed and compared with the experiment data to obtain the material properties. Simulation setup about the inlet and outlet pressure follows the setup in the experiment. Simulation results including the fundamental frequency, air flow rate, the divergent angle will be compared with the experimental data, providing the validation of the simulation approach. The relationship between flow separation, intra-glottal vortices, divergent angle and flow rate will be comprehensively analyzed.

Effect of pH on the hinge region of influenza viral protein: a combined constant pH and well-tempered molecular dynamics study

NASA Astrophysics Data System (ADS)

Pathak, Arup Kumar

2018-05-01

Despite the knowledge that the influenza protein, hemagglutinin, undergoes a large conformational change at low pH during the process of fusion with the host cell, its molecular mechanism remains elusive. The present constant pH molecular dynamics (CpHMD) study identifies the residues responsible for large conformational change in acidic condition. Based on the pKa calculations, it is predicted that His-106 is much more responsible for the large conformational change than any other residues in the hinge region of hemagglutinin protein. Potential of mean force profile from well-tempered meta-dynamics (WT-MtD) simulation is also generated along the folding pathway by considering radius of gyration (R gyr) as a collective variable (CV). It is very clear from the present WT-MtD study, that the initial bending starts at that hinge region, which may trigger other conformational changes. Both the protein–protein and protein–water HB time correlation functions are monitored along the folding pathway. The protein–protein (full or hinge region) HB time correlation functions are always found to be stronger than those of the protein–water time correlation functions. The dynamical balance between protein–protein and protein–water HB interactions favors the stabilization of the folded state.

A folding-dependent mechanism of antimicrobial peptide resistance to degradation unveiled by solution structure of distinctin

PubMed Central

Raimondo, Domenico; Andreotti, Giuseppina; Saint, Nathalie; Amodeo, Pietro; Renzone, Giovanni; Sanseverino, Marina; Zocchi, Ivana; Molle, Gerard; Motta, Andrea; Scaloni, Andrea

2005-01-01

Many bioactive peptides, presenting an unstructured conformation in aqueous solution, are made resistant to degradation by posttranslational modifications. Here, we describe how molecular oligomerization in aqueous solution can generate a still unknown transport form for amphipathic peptides, which is more compact and resistant to proteases than forms related to any possible monomer. This phenomenon emerged from 3D structure, function, and degradation properties of distinctin, a heterodimeric antimicrobial compound consisting of two peptide chains linked by a disulfide bond. After homodimerization in water, this peptide exhibited a fold consisting of a symmetrical full-parallel four-helix bundle, with a well secluded hydrophobic core and exposed basic residues. This fold significantly stabilizes distinctin against proteases compared with other linear amphipathic peptides, without affecting its antimicrobial, hemolytic, and ion-channel formation properties after membrane interaction. This full-parallel helical orientation represents a perfect compromise between formation of a stable structure in water and requirement of a drastic structural rearrangement in membranes to elicit antimicrobial potential. Thus, distinctin can be claimed as a prototype of a previously unrecognized class of antimicrobial derivatives. These results suggest a critical revision of the role of peptide oligomerization whenever solubility or resistance to proteases is known to affect biological properties. PMID:15840728

Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules.

PubMed

Zhao, Tao; Liu, Ran; Ding, Xiaofan; Zhao, Juncai; Yu, Haixiang; Wang, Lei; Xu, Qing; Wang, Xuan; Lou, Xinhui; He, Miao; Xiao, Yi

2015-08-04

It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000-100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms.

Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues† †Electronic supplementary information (ESI) available: Synthesis of TEG–BA, Gluen–BA, mGluen–BA and Gluen–Ph; 1H NMR, 13C NMR, MALDI-TOF MS, electronic absorption, and CD spectra; zeta potential distributions; SLS plots; DLS histograms; and related experimental procedures. See DOI: 10.1039/c5sc00524h Click here for additional data file.

PubMed Central

Mogaki, Rina

2015-01-01

Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Gluen–BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Gluen–BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10–BA and Glue29–BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG–BA). Most importantly, Glue10–BA inhibited the protease activity of trypsin 13-fold more than TEG–BA. In sharp contrast, mGlue27–BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG–BA for trypsin, was inferior even to TEG–BA for trypsin inhibition. PMID:28706668

Streptococcus mutans dextransucrase: stimulation by phospholipids from human sera and oral fluids.

PubMed Central

Schachtele, C F; Harlander, S K; Bracke, J W; Ostrum, L C; Maltais, J A; Billings, R J

1978-01-01

Serum, gingival crevicular fluid, and parotid, submandibular, and labial minor gland saliva from four individuals stimulated glucan formation from sucrose by the Streptococcus mutans strain 6715 dextransucrase (EC 2.4.1.5). At final dilutions of 1:10 all of the fluids stimulated crude enzyme preparations approximately 1.8-fold. The fluids stimulated the purified water-insoluble glucan-synthesizing form of the dextransucrase approximately 3.2-fold and the water-soluble glucan-producing form of the enzyme approximately 2.4-fold. The fluids all contained concentrations of stimulatory material that could be reduced to undetectable levels only after dilutions of greater than 1:1,000. The increased rates of glucan formation caused by the fluids and dextran were additive, indicating that stimulation by the fluids was primarily due to interactions with entities other than glucan primer molecules. In contrast, the elevated levels of glucan formation in the presence of the fluids was not further enhanced by the addition of lysophosphatidylcholine. Lysophosphatidylcholine purified from parotid and submandibular saliva by solvent extraction and thin-layer chromatography stimulated the dextransucrase as effectively as egg yolk lysophosphatidylcholine. Thus, phospholipids normally found in human oral fluids can enhance the activity of an enzyme believed to be directly associated with the cariogenic potential of S. mutans. PMID:365766

Folding thermodynamics of model four-strand antiparallel beta-sheet proteins.

PubMed Central

Jang, Hyunbum; Hall, Carol K; Zhou, Yaoqi

2002-01-01

The thermodynamic properties for three different types of off-lattice four-strand antiparallel beta-strand protein models interacting via a hybrid Go-type potential have been investigated. Discontinuous molecular dynamic simulations have been performed for different sizes of the bias gap g, an artificial measure of a model protein's preference for its native state. The thermodynamic transition temperatures are obtained by calculating the squared radius of gyration R(g)(2), the root-mean-squared pair separation fluctuation Delta(B), the specific heat C(v), the internal energy of the system E, and the Lindemann disorder parameter Delta(L). Despite these models' simplicity, they exhibit a complex set of protein transitions, consistent with those observed in experimental studies on real proteins. Starting from high temperature, these transitions include a collapse transition, a disordered-to-ordered globule transition, a folding transition, and a liquid-to-solid transition. The high temperature transitions, i.e., the collapse transition and the disordered-to-ordered globule transition, exist for all three beta-strand proteins, although the native-state geometry of the three model proteins is different. However the low temperature transitions, i.e., the folding transition and the liquid-to-solid transition, strongly depend on the native-state geometry of the model proteins and the size of the bias gap. PMID:11806908

High-Performance Agent-Based Modeling Applied to Vocal Fold Inflammation and Repair.

PubMed

Seekhao, Nuttiiya; Shung, Caroline; JaJa, Joseph; Mongeau, Luc; Li-Jessen, Nicole Y K

2018-01-01

Fast and accurate computational biology models offer the prospect of accelerating the development of personalized medicine. A tool capable of estimating treatment success can help prevent unnecessary and costly treatments and potential harmful side effects. A novel high-performance Agent-Based Model (ABM) was adopted to simulate and visualize multi-scale complex biological processes arising in vocal fold inflammation and repair. The computational scheme was designed to organize the 3D ABM sub-tasks to fully utilize the resources available on current heterogeneous platforms consisting of multi-core CPUs and many-core GPUs. Subtasks are further parallelized and convolution-based diffusion is used to enhance the performance of the ABM simulation. The scheme was implemented using a client-server protocol allowing the results of each iteration to be analyzed and visualized on the server (i.e., in-situ ) while the simulation is running on the same server. The resulting simulation and visualization software enables users to interact with and steer the course of the simulation in real-time as needed. This high-resolution 3D ABM framework was used for a case study of surgical vocal fold injury and repair. The new framework is capable of completing the simulation, visualization and remote result delivery in under 7 s per iteration, where each iteration of the simulation represents 30 min in the real world. The case study model was simulated at the physiological scale of a human vocal fold. This simulation tracks 17 million biological cells as well as a total of 1.7 billion signaling chemical and structural protein data points. The visualization component processes and renders all simulated biological cells and 154 million signaling chemical data points. The proposed high-performance 3D ABM was verified through comparisons with empirical vocal fold data. Representative trends of biomarker predictions in surgically injured vocal folds were observed.

High-Performance Agent-Based Modeling Applied to Vocal Fold Inflammation and Repair

PubMed Central

Seekhao, Nuttiiya; Shung, Caroline; JaJa, Joseph; Mongeau, Luc; Li-Jessen, Nicole Y. K.

2018-01-01

Fast and accurate computational biology models offer the prospect of accelerating the development of personalized medicine. A tool capable of estimating treatment success can help prevent unnecessary and costly treatments and potential harmful side effects. A novel high-performance Agent-Based Model (ABM) was adopted to simulate and visualize multi-scale complex biological processes arising in vocal fold inflammation and repair. The computational scheme was designed to organize the 3D ABM sub-tasks to fully utilize the resources available on current heterogeneous platforms consisting of multi-core CPUs and many-core GPUs. Subtasks are further parallelized and convolution-based diffusion is used to enhance the performance of the ABM simulation. The scheme was implemented using a client-server protocol allowing the results of each iteration to be analyzed and visualized on the server (i.e., in-situ) while the simulation is running on the same server. The resulting simulation and visualization software enables users to interact with and steer the course of the simulation in real-time as needed. This high-resolution 3D ABM framework was used for a case study of surgical vocal fold injury and repair. The new framework is capable of completing the simulation, visualization and remote result delivery in under 7 s per iteration, where each iteration of the simulation represents 30 min in the real world. The case study model was simulated at the physiological scale of a human vocal fold. This simulation tracks 17 million biological cells as well as a total of 1.7 billion signaling chemical and structural protein data points. The visualization component processes and renders all simulated biological cells and 154 million signaling chemical data points. The proposed high-performance 3D ABM was verified through comparisons with empirical vocal fold data. Representative trends of biomarker predictions in surgically injured vocal folds were observed. PMID:29706894

Current cytochrome P450 phenotyping methods applied to metabolic drug-drug interaction prediction in dogs.

PubMed

Mills, Beth Miskimins; Zaya, Matthew J; Walters, Rodney R; Feenstra, Kenneth L; White, Julie A; Gagne, Jason; Locuson, Charles W

2010-03-01

Recombinant cytochrome P450 (P450) phenotyping, different approaches for estimating fraction metabolized (f(m)), and multiple measures of in vivo inhibitor exposure were tested for their ability to predict drug interaction magnitude in dogs. In previous reports, midazolam-ketoconazole interaction studies in dogs have been attributed to inhibition of CYP3A pathways. However, in vitro phenotyping studies demonstrated higher apparent intrinsic clearances (CL(int,app)) of midazolam with canine CYP2B11 and CYP2C21. Application of activity correction factors and isoform hepatic abundance to liver microsome CL(int,app) values further implicated CYP2B11 (f(m) >or= 0.89) as the dog enzyme responsible for midazolam- and temazepam-ketoconazole interactions in vivo. Mean area under the curve (AUC) in the presence of the inhibitor/AUC ratios from intravenous and oral midazolam interaction studies were predicted well with unbound K(i) and estimates of unbound hepatic inlet inhibitor concentrations and intestinal metabolism using the AUC-competitive inhibitor relationship. No interactions were observed in vivo with bufuralol, although significant interactions with bufuralol were predicted with fluoxetine via CYP2D and CYP2C pathways (>2.45-fold) but not with clomipramine (<2-fold). The minor caffeine-fluvoxamine interaction (1.78-fold) was slightly higher than predicted values based on determination of a moderate f(m) value for CYP1A1, although CYP1A2 may also be involved in caffeine metabolism. The findings suggest promise for in vitro approaches to drug interaction assessment in dogs, but they also highlight the need to identify improved substrate and inhibitor probes for canine P450s.

Structures of p -shell double-Λ hypernuclei studied with microscopic cluster models

NASA Astrophysics Data System (ADS)

Kanada-En'yo, Yoshiko

2018-03-01

0 s -orbit Λ states in p -shell double-Λ hypernuclei (Z Λ Λ A ), Li Λ Λ 8 , Li Λ Λ 9 , Be Λ Λ 10 ,11 ,12 , B Λ Λ 12 ,13 , and C Λ Λ 14 are investigated. Microscopic cluster models are applied to core nuclear part and a potential model is adopted for Λ particles. The Λ -core potential is a folding potential obtained with effective G -matrix Λ -N interactions, which reasonably reproduce energy spectra of Z Λ A -1 . System dependence of the Λ -Λ binding energies is understood by the core polarization energy from nuclear size reduction. Reductions of nuclear sizes and E 2 transition strengths by Λ particles are also discussed.

Computing Prediction and Functional Analysis of Prokaryotic Propionylation.

PubMed

Wang, Li-Na; Shi, Shao-Ping; Wen, Ping-Ping; Zhou, Zhi-You; Qiu, Jian-Ding

2017-11-27

Identification and systematic analysis of candidates for protein propionylation are crucial steps for understanding its molecular mechanisms and biological functions. Although several proteome-scale methods have been performed to delineate potential propionylated proteins, the majority of lysine-propionylated substrates and their role in pathological physiology still remain largely unknown. By gathering various databases and literatures, experimental prokaryotic propionylation data were collated to be trained in a support vector machine with various features via a three-step feature selection method. A novel online tool for seeking potential lysine-propionylated sites (PropSeek) ( http://bioinfo.ncu.edu.cn/PropSeek.aspx ) was built. Independent test results of leave-one-out and n-fold cross-validation were similar to each other, showing that PropSeek is a stable and robust predictor with satisfying performance. Meanwhile, analyses of Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathways, and protein-protein interactions implied a potential role of prokaryotic propionylation in protein synthesis and metabolism.

The Role of High-Dimensional Diffusive Search, Stabilization, and Frustration in Protein Folding

PubMed Central

Rimratchada, Supreecha; McLeish, Tom C.B.; Radford, Sheena E.; Paci, Emanuele

2014-01-01

Proteins are polymeric molecules with many degrees of conformational freedom whose internal energetic interactions are typically screened to small distances. Therefore, in the high-dimensional conformation space of a protein, the energy landscape is locally relatively flat, in contrast to low-dimensional representations, where, because of the induced entropic contribution to the full free energy, it appears funnel-like. Proteins explore the conformation space by searching these flat subspaces to find a narrow energetic alley that we call a hypergutter and then explore the next, lower-dimensional, subspace. Such a framework provides an effective representation of the energy landscape and folding kinetics that does justice to the essential characteristic of high-dimensionality of the search-space. It also illuminates the important role of nonnative interactions in defining folding pathways. This principle is here illustrated using a coarse-grained model of a family of three-helix bundle proteins whose conformations, once secondary structure has formed, can be defined by six rotational degrees of freedom. Two folding mechanisms are possible, one of which involves an intermediate. The stabilization of intermediate subspaces (or states in low-dimensional projection) in protein folding can either speed up or slow down the folding rate depending on the amount of native and nonnative contacts made in those subspaces. The folding rate increases due to reduced-dimension pathways arising from the mere presence of intermediate states, but decreases if the contacts in the intermediate are very stable and introduce sizeable topological or energetic frustration that needs to be overcome. Remarkably, the hypergutter framework, although depending on just a few physically meaningful parameters, can reproduce all the types of experimentally observed curvature in chevron plots for realizations of this fold. PMID:24739172

The prediction of drug metabolism, tissue distribution, and bioavailability of 50 structurally diverse compounds in rat using mechanism-based absorption, distribution, and metabolism prediction tools.

PubMed

De Buck, Stefan S; Sinha, Vikash K; Fenu, Luca A; Gilissen, Ron A; Mackie, Claire E; Nijsen, Marjoleen J

2007-04-01

The aim of this study was to assess a physiologically based modeling approach for predicting drug metabolism, tissue distribution, and bioavailability in rat for a structurally diverse set of neutral and moderate-to-strong basic compounds (n = 50). Hepatic blood clearance (CL(h)) was projected using microsomal data and shown to be well predicted, irrespective of the type of hepatic extraction model (80% within 2-fold). Best predictions of CL(h) were obtained disregarding both plasma and microsomal protein binding, whereas strong bias was seen using either blood binding only or both plasma and microsomal protein binding. Two mechanistic tissue composition-based equations were evaluated for predicting volume of distribution (V(dss)) and tissue-to-plasma partitioning (P(tp)). A first approach, which accounted for ionic interactions with acidic phospholipids, resulted in accurate predictions of V(dss) (80% within 2-fold). In contrast, a second approach, which disregarded ionic interactions, was a poor predictor of V(dss) (60% within 2-fold). The first approach also yielded accurate predictions of P(tp) in muscle, heart, and kidney (80% within 3-fold), whereas in lung, liver, and brain, predictions ranged from 47% to 62% within 3-fold. Using the second approach, P(tp) prediction accuracy in muscle, heart, and kidney was on average 70% within 3-fold, and ranged from 24% to 54% in all other tissues. Combining all methods for predicting V(dss) and CL(h) resulted in accurate predictions of the in vivo half-life (70% within 2-fold). Oral bioavailability was well predicted using CL(h) data and Gastroplus Software (80% within 2-fold). These results illustrate that physiologically based prediction tools can provide accurate predictions of rat pharmacokinetics.

Magnetic micro/nanoparticle flocculation-based signal amplification for biosensing

PubMed Central

Mzava, Omary; Taş, Zehra; İçöz, Kutay

2016-01-01

We report a time and cost efficient signal amplification method for biosensors employing magnetic particles. In this method, magnetic particles in an applied external magnetic field form magnetic dipoles, interact with each other, and accumulate along the magnetic field lines. This magnetic interaction does not need any biomolecular coating for binding and can be controlled with the strength of the applied magnetic field. The accumulation can be used to amplify the corresponding pixel area that is obtained from an image of a single magnetic particle. An application of the method to the Escherichia coli 0157:H7 bacteria samples is demonstrated in order to show the potential of the approach. A minimum of threefold to a maximum of 60-fold amplification is reached from a single bacteria cell under a magnetic field of 20 mT. PMID:27354793

Transiently disordered tails accelerate folding of globular proteins.

PubMed

Mallik, Saurav; Ray, Tanaya; Kundu, Sudip

2017-07-01

Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (TstDREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at TstDREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of TstDREFs in shaping the folding kinetics. TstDREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by TstDREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the TstDREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing. © 2017 Federation of European Biochemical Societies.

GIMDA: Graphlet interaction-based MiRNA-disease association prediction.

PubMed

Chen, Xing; Guan, Na-Na; Li, Jian-Qiang; Yan, Gui-Ying

2018-03-01

MicroRNAs (miRNAs) have been confirmed to be closely related to various human complex diseases by many experimental studies. It is necessary and valuable to develop powerful and effective computational models to predict potential associations between miRNAs and diseases. In this work, we presented a prediction model of Graphlet Interaction for MiRNA-Disease Association prediction (GIMDA) by integrating the disease semantic similarity, miRNA functional similarity, Gaussian interaction profile kernel similarity and the experimentally confirmed miRNA-disease associations. The related score of a miRNA to a disease was calculated by measuring the graphlet interactions between two miRNAs or two diseases. The novelty of GIMDA lies in that we used graphlet interaction to analyse the complex relationships between two nodes in a graph. The AUCs of GIMDA in global and local leave-one-out cross-validation (LOOCV) turned out to be 0.9006 and 0.8455, respectively. The average result of five-fold cross-validation reached to 0.8927 ± 0.0012. In case study for colon neoplasms, kidney neoplasms and prostate neoplasms based on the database of HMDD V2.0, 45, 45, 41 of the top 50 potential miRNAs predicted by GIMDA were validated by dbDEMC and miR2Disease. Additionally, in the case study of new diseases without any known associated miRNAs and the case study of predicting potential miRNA-disease associations using HMDD V1.0, there were also high percentages of top 50 miRNAs verified by the experimental literatures. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Simple physics-based analytical formulas for the potentials of mean force of the interaction of amino-acid side chains in water. V. Like-charged side chains.

PubMed

Makowski, Mariusz; Liwo, Adam; Sobolewski, Emil; Scheraga, Harold A

2011-05-19

A new model of side-chain-side-chain interactions for charged side-chains of amino acids, to be used in the UNRES force-field, has been developed, in which a side chain consists of a nonpolar and a charged site. The interaction energy between the nonpolar sites is composed of a Gay-Berne and a cavity term; the interaction energy between the charged sites consists of a Lennard-Jones term, a Coulombic term, a generalized-Born term, and a cavity term, while the interaction energy between the nonpolar and charged sites is composed of a Gay-Berne and a polarization term. We parametrized the energy function for the models of all six pairs of natural like-charged amino-acid side chains, namely propionate-propionate (for the aspartic acid-aspartic acid pair), butyrate-butyrate (for the glutamic acid-glutamic acid pair), propionate-butyrate (for the aspartic acid-glutamic acid pair), pentylamine cation-pentylamine cation (for the lysine-lysine pair), 1-butylguanidine cation-1-butylguanidine cation (for the arginine-arginine pair), and pentylamine cation-1-butylguanidine cation (for the lysine-arginine pair). By using umbrella-sampling molecular dynamics simulations in explicit TIP3P water, we determined the potentials of mean force of the above-mentioned pairs as functions of distance and orientation and fitted analytical expressions to them. The positions and depths of the contact minima and the positions and heights of the desolvation maxima, including their dependence on the orientation of the molecules were well represented by analytical expressions for all systems. The values of the parameters of all the energy components are physically reasonable, which justifies use of such potentials in coarse-grain protein-folding simulations. © 2011 American Chemical Society

When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

PubMed

Muñoz, Victor; Cerminara, Michele

2016-09-01

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).

Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction

NASA Astrophysics Data System (ADS)

Lu, Yi-Ching; Luo, Pei-Chun; Huang, Chun-Wan; Leu, Yann-Lii; Wang, Tzu-Hao; Wei, Kuo-Chen; Wang, Hsin-Ell; Ma, Yunn-Hwa

2014-08-01

Nanoparticles may serve as carriers in targeted therapeutics; interaction of the nanoparticles with a biological system may determine their targeting effects and therapeutic efficacy. Epigallocatechin-3-gallate (EGCG), a major component of tea catechins, has been conjugated with nanoparticles and tested as an anticancer agent. We investigated whether EGCG may enhance nanoparticle uptake by tumor cells. Cellular uptake of a dextran-coated magnetic nanoparticle (MNP) was determined by confocal microscopy, flow cytometry or a potassium thiocyanate colorimetric method. We demonstrated that EGCG greatly enhanced interaction and/or internalization of MNPs (with or without polyethylene glycol) by glioma cells, but not vascular endothelial cells. The enhancing effects are both time- and concentration-dependent. Such effects may be induced by a simple mix of MNPs with EGCG at a concentration as low as 1-3 μM, which increased MNP uptake 2- to 7-fold. In addition, application of magnetic force further potentiated MNP uptake, suggesting a synergetic effect of EGCG and magnetic force. Because the effects of EGCG were preserved at 4 °C, but not when EGCG was removed from the culture medium prior to addition of MNPs, a direct interaction of EGCG and MNPs was implicated. Use of an MNP-EGCG composite produced by adsorption of EGCG and magnetic separation also led to an enhanced uptake. The results reveal a novel interaction of a food component and nanocarrier system, which may be potentially amenable to magnetofection, cell labeling/tracing, and targeted therapeutics.

Interplay between binding affinity and kinetics in protein-protein interactions.

PubMed

Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

2016-07-01

To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cross-interactions between the Alzheimer Disease Amyloid-β Peptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis.

PubMed

Luo, Jinghui; Wärmländer, Sebastian K T S; Gräslund, Astrid; Abrahams, Jan Pieter

2016-08-05

Many protein folding diseases are intimately associated with accumulation of amyloid aggregates. The amyloid materials formed by different proteins/peptides share many structural similarities, despite sometimes large amino acid sequence differences. Some amyloid diseases constitute risk factors for others, and the progression of one amyloid disease may affect the progression of another. These connections are arguably related to amyloid aggregates of one protein being able to directly nucleate amyloid formation of another, different protein: the amyloid cross-interaction. Here, we discuss such cross-interactions between the Alzheimer disease amyloid-β (Aβ) peptide and other amyloid proteins in the context of what is known from in vitro and in vivo experiments, and of what might be learned from clinical studies. The aim is to clarify potential molecular associations between different amyloid diseases. We argue that the amyloid cascade hypothesis in Alzheimer disease should be expanded to include cross-interactions between Aβ and other amyloid proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein–Mineral Interactions: Molecular Dynamics Simulations Capture Importance of Variations in Mineral Surface Composition and Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, Amity; Reardon, Patrick N.; Chacon, Stephany S.

Molecular dynamics simulations, conventional and metadynamics, were performed to determine the interaction of model protein Gb1 over kaolinite (001), Na+-montmorillonite (001), Ca2+-montmorillonite (001), goethite (100), and Na+-birnessite (001) mineral surfaces. Gb1, a small (56 residue) protein with a well-characterized solution-state nuclear magnetic resonance (NMR) structure and having α-helix, four-fold β-sheet, and hydrophobic core features, is used as a model protein to study protein soil mineral interactions and gain insights on structural changes and potential degradation of protein. From our simulations, we observe little change to the hydrated Gb1 structure over the kaolinite, montmorillonite, and goethite surfaces relative to its solvatedmore » structure without these mineral surfaces present. Over the Na+-birnessite basal surface, however, the Gb1 structure is highly disturbed as a result of interaction with this birnessite surface. Unraveling of the Gb1 β-sheet at specific turns and a partial unraveling of the α-helix is observed over birnessite, which suggests specific vulnerable residue sites for oxidation or hydrolysis possibly leading to fragmentation.« less

Mechanism of hard-nanomaterial clearance by the liver.

PubMed

Tsoi, Kim M; MacParland, Sonya A; Ma, Xue-Zhong; Spetzler, Vinzent N; Echeverri, Juan; Ouyang, Ben; Fadel, Saleh M; Sykes, Edward A; Goldaracena, Nicolas; Kaths, Johann M; Conneely, John B; Alman, Benjamin A; Selzner, Markus; Ostrowski, Mario A; Adeyi, Oyedele A; Zilman, Anton; McGilvray, Ian D; Chan, Warren C W

2016-11-01

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.

Exact solution of a one-dimensional model of strained epitaxy on a periodically modulated substrate

NASA Astrophysics Data System (ADS)

Tokar, V. I.; Dreyssé, H.

2005-03-01

We consider a one-dimensional lattice gas model of strained epitaxy with the elastic strain accounted for through a finite number of cluster interactions comprising contiguous atomic chains. Interactions of this type arise in the models of strained epitaxy based on the Frenkel-Kontorova model. Furthermore, the deposited atoms interact with the substrate via an arbitrary periodic potential of period L . This model is solved exactly with the use of an appropriately adopted technique developed recently in the theory of protein folding. The advantage of the proposed approach over the standard transfer-matrix method is that it reduces the problem to finding the largest eigenvalue of a matrix of size L instead of 2L-1 , which is vital in the case of nanostructures where L may measure in hundreds of interatomic distances. Our major conclusion is that the substrate modulation always facilitates the size calibration of self-assembled nanoparticles in one- and two-dimensional systems.

Fluid-acoustic interactions and their impact on pathological voiced speech

NASA Astrophysics Data System (ADS)

Erath, Byron D.; Zanartu, Matias; Peterson, Sean D.; Plesniak, Michael W.

2011-11-01

Voiced speech is produced by vibration of the vocal fold structures. Vocal fold dynamics arise from aerodynamic pressure loadings, tissue properties, and acoustic modulation of the driving pressures. Recent speech science advancements have produced a physiologically-realistic fluid flow solver (BLEAP) capable of prescribing asymmetric intraglottal flow attachment that can be easily assimilated into reduced order models of speech. The BLEAP flow solver is extended to incorporate acoustic loading and sound propagation in the vocal tract by implementing a wave reflection analog approach for sound propagation based on the governing BLEAP equations. This enhanced physiological description of the physics of voiced speech is implemented into a two-mass model of speech. The impact of fluid-acoustic interactions on vocal fold dynamics is elucidated for both normal and pathological speech through linear and nonlinear analysis techniques. Supported by NSF Grant CBET-1036280.

Cocaine-like discriminative stimulus effects of "norepinephrine-preferring" monoamine releasers: time course and interaction studies in rhesus monkeys.

PubMed

Kohut, Stephen J; Jacobs, David S; Rothman, Richard B; Partilla, John S; Bergman, Jack; Blough, Bruce E

2017-12-01

The therapeutic potential of monoamine releasers with prominent dopaminergic effects is hindered by their high abuse liability. The present study examined the effects of several novel "norepinephrine (NE)-preferring" monoamine releasers relative to non-selective monoamine releasers, d-amphetamine and d-methamphetamine, in rhesus monkeys trained to discriminate cocaine. NE-preferring releasers were approximately 13-fold more potent for NE compared to dopamine release and ranged in potency for serotonin release (PAL-329 < l-methamphetamine < PAL-169). Adult rhesus macaques were trained to discriminate 0.4 mg/kg, IM cocaine on a 30-response fixed ratio schedule of food reinforcement. Substitution studies determined the extent to which test drugs produced cocaine-like discriminative stimulus effects and their time course. Drug interaction studies determined whether pretreatment with test drugs altered the discriminable effects of cocaine. Results show that cocaine, d-amphetamine, and d-methamphetamine dose-dependently substituted for cocaine with similar potencies. Among the "NE-preferring" releasers, PAL-329 and l-methamphetamine also dose-dependently substituted for cocaine but differed in potency. PAL-169 failed to substitute for cocaine up to a dose that disrupted responding. When administered prior to cocaine, only d-amphetamine and PAL-329 significantly shifted the cocaine dose-effect function leftward indicating enhancement of cocaine's discriminative stimulus effects. These data suggest that greater potency for NE relative to dopamine release (up to 13-fold) does not interfere with the ability of a monoamine releaser to produce cocaine-like discriminative effects but that increased serotonin release may have an inhibitory effect. Further characterization of these and other "NE-preferring" monoamine releasers should provide insight into their potential for the management of cocaine addiction.

Phenobarbital Induction and Chemical Synergism Demonstrate the Role of UDP-Glucuronosyltransferases in Detoxification of Naphthalophos by Haemonchus contortus Larvae

PubMed Central

Ruffell, Angela P.; Ingham, Aaron B.

2014-01-01

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field. PMID:25288079

Phenobarbital induction and chemical synergism demonstrate the role of UDP-glucuronosyltransferases in detoxification of naphthalophos by Haemonchus contortus larvae.

PubMed

Kotze, Andrew C; Ruffell, Angela P; Ingham, Aaron B

2014-12-01

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

BEL β-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms.

PubMed

Bovi, Michele; Cenci, Lucia; Perduca, Massimiliano; Capaldi, Stefano; Carrizo, Maria E; Civiero, Laura; Chiarelli, Laurent R; Galliano, Monica; Monaco, Hugo L

2013-05-01

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

Tackling force-field bias in protein folding simulations: folding of Villin HP35 and Pin WW domains in explicit water.

PubMed

Mittal, Jeetain; Best, Robert B

2010-08-04

The ability to fold proteins on a computer has highlighted the fact that existing force fields tend to be biased toward a particular type of secondary structure. Consequently, force fields for folding simulations are often chosen according to the native structure, implying that they are not truly "transferable." Here we show that, while the AMBER ff03 potential is known to favor helical structures, a simple correction to the backbone potential (ff03( *)) results in an unbiased energy function. We take as examples the 35-residue alpha-helical Villin HP35 and 37 residue beta-sheet Pin WW domains, which had not previously been folded with the same force field. Starting from unfolded configurations, simulations of both proteins in Amber ff03( *) in explicit solvent fold to within 2.0 A RMSD of the experimental structures. This demonstrates that a simple backbone correction results in a more transferable force field, an important requirement if simulations are to be used to interpret folding mechanism. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The role of hepatic transport and metabolism in the interactions between pravastatin or repaglinide and two rOatp inhibitors in rats.

PubMed

Badolo, Lassina; Bundgaard, Christoffer; Garmer, Mats; Jensen, Bente

2013-07-16

A change in the function or expression of hepatic drug transporters may have significant effect on the efficacy or safety of orally administered drugs. Although a number of clinical drug-drug interactions associated with hepatic transport proteins have been reported, in practice it is not always straightforward to discriminate other pathways (e.g. drug metabolism) from being involved in these interactions. The present study was designed to assess the interactions between organic anion transporting polypeptide (Oatp) substrates (pravastatin or repaglinide) and inhibitors (spironolactone or diphenhydramine) in vivo in rats. The mechanisms behind the interactions were then investigated using in vitro tools (isolated hepatocytes and rat liver microsomes). The results showed a significant increase in the systemic exposures of pravastatin (2.5-fold increase in AUC) and repaglinide (1.8-fold increase in AUC) after co-administration of spironolactone to rats. Diphenhydramine increased the AUC of repaglinide by 1.4-fold. The in vivo interactions observed in rats between Oatp substrates and inhibitors may a priori be classified as transport-mediated drug-drug interactions. However, mechanistic studies performed in vitro using both isolated rat hepatocytes and rat liver microsomes showed that the interaction between pravastatin and spironolactone may be solely linked to the inhibition of pravastatin uptake in liver. On the contrary, the inhibition of cytochrome P450 seemed to be the reason for the interactions observed between repaglinide and spironolactone. Although the function and structure of transport proteins may vary between rats and humans, the approach used in the present study can be applied to humans and help to understand the role of drug transport and drug metabolism in a given drug-drug interaction. This is important to predict and mitigate the risk of drug-drug interactions for a candidate drug in pre-clinical development, it is also important for the optimal design of drug-drug interactions studies in the clinic. Copyright © 2013 Elsevier B.V. All rights reserved.

Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

PubMed Central

Bose, Sayantan; Heath, Carissa M.; Shah, Priya A.; Alayyoubi, Maher; Jardetzky, Theodore S.

2013-01-01

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger. PMID:24089572

Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.

PubMed

Vuillard, L; Rabilloud, T; Goldberg, M E

1998-08-15

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

Numerical solution of fluid-structure interaction represented by human vocal folds in airflow

NASA Astrophysics Data System (ADS)

Valášek, J.; Sváček, P.; Horáček, J.

2016-03-01

The paper deals with the human vocal folds vibration excited by the fluid flow. The vocal fold is modelled as an elastic body assuming small displacements and therefore linear elasticity theory is used. The viscous incompressible fluid flow is considered. For purpose of numerical solution the arbitrary Lagrangian-Euler method (ALE) is used. The whole problem is solved by the finite element method (FEM) based solver. Results of numerical experiments with different boundary conditions are presented.

Cloning and Expression of β-Defensin from Soiny Mullet (Liza haematocheila), with Insights of its Antibacterial Mechanism

PubMed Central

Qi, Zhitao; Xu, Wei; Meng, Fancui; Zhang, Qihuan; Chen, Chenglung; Shao, Rong

2016-01-01

Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the β-defensin (Lhβ-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhβ-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhβ-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) β-defensin. Realtime PCR revealed that Lhβ-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhβ-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhβ-defensin possessed a monomeric structure of a three-stranded anti-parallel β-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of β-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhβ-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel β1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two β1-strands. The most important interactions between β-defensin and membrane are the electrostatic interactions between Arg residues in β-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish β-defensins. PMID:27322675

Solvent Effects on Protein Folding/Unfolding

NASA Astrophysics Data System (ADS)

García, A. E.; Hillson, N.; Onuchic, J. N.

Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.

Arginine analogues incorporating carboxylate bioisosteric functions are micromolar inhibitors of human recombinant DDAH-1.

PubMed

Tommasi, Sara; Zanato, Chiara; Lewis, Benjamin C; Nair, Pramod C; Dall'Angelo, Sergio; Zanda, Matteo; Mangoni, Arduino A

2015-12-14

Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme involved in the metabolism of asymmetric dimethylarginine (ADMA) and N-monomethyl arginine (NMMA), which are endogenous inhibitors of the nitric oxide synthase (NOS) family of enzymes. Two isoforms of DDAH have been identified in humans, DDAH-1 and DDAH-2. DDAH-1 inhibition represents a promising strategy to limit the overproduction of NO in pathological states without affecting the homeostatic role of this important messenger molecule. Here we describe the design and synthesis of 12 novel DDAH-1 inhibitors and report their derived kinetic parameters, IC50 and Ki. Arginine analogue 10a, characterized by an acylsulfonamide isosteric replacement of the carboxylate, showed a 13-fold greater inhibitory potential relative to the known DDAH-1 inhibitor, L-257. Compound 10a was utilized to study the putative binding interactions of human DDAH-1 inhibition using molecular dynamics simulations. The latter suggests that several stabilizing interactions occur in the DDAH-1 active-site, providing structural insights for the enhanced inhibitory potential demonstrated by in vitro inhibition studies.

The Role of Non-Native Interactions in the Folding of Knotted Proteins: Insights from Molecular Dynamics Simulations

PubMed Central

Covino, Roberto; Škrbić, Tatjana; Beccara, Silvio a; Faccioli, Pietro; Micheletti, Cristian

2014-01-01

For several decades, the presence of knots in naturally-occurring proteins was largely ruled out a priori for its supposed incompatibility with the efficiency and robustness of folding processes. For this very same reason, the later discovery of several unrelated families of knotted proteins motivated researchers to look into the physico-chemical mechanisms governing the concerted sequence of folding steps leading to the consistent formation of the same knot type in the same protein location. Besides experiments, computational studies are providing considerable insight into these mechanisms. Here, we revisit a number of such recent investigations within a common conceptual and methodological framework. By considering studies employing protein models with different structural resolution (coarse-grained or atomistic) and various force fields (from pure native-centric to realistic atomistic ones), we focus on the role of native and non-native interactions. For various unrelated instances of knotted proteins, non-native interactions are shown to be very important for favoring the emergence of conformations primed for successful self-knotting events. PMID:24970203

Fluoxetine and norfluoxetine mediated complex drug-drug interactions: in vitro to in vivo correlation of effects on CYP2D6, CYP2C19 and CYP3A4

PubMed Central

Sager, Jennifer E; Lutz, Justin D; Foti, Robert S; Davis, Connie; Kunze, Kent L; Isoherranen, Nina

2014-01-01

Fluoxetine and its circulating metabolite norfluoxetine present a complex multiple inhibitor system that causes reversible or time-dependent inhibition of CYP2D6, CYP3A4, and CYP2C19 in vitro. While significant inhibition of all three enzymes in vivo is predicted, midazolam and lovastatin AUCs were unaffected by two week dosing of fluoxetine whereas dextromethorphan AUC was increased by 27-fold and omeprazole AUC by 7.1-fold. This observed discrepancy between in vitro risk assessment and in vivo DDI profile was rationalized by time-varying dynamic pharmacokinetic models that incorporated circulating concentrations of fluoxetine and norfluoxetine enantiomers, mutual inhibitor-inhibitor interactions and CYP3A4 induction. The dynamic models predicted all DDIs with less than 2-fold error. This study demonstrates that complex drug-drug interactions that involve multiple mechanisms, pathways and inhibitors with their metabolites can be predicted and rationalized via characterization of all the inhibitory species in vitro. PMID:24569517

Reduced atomic pair-interaction design (RAPID) model for simulations of proteins.

PubMed

Ni, Boris; Baumketner, Andrij

2013-02-14

Increasingly, theoretical studies of proteins focus on large systems. This trend demands the development of computational models that are fast, to overcome the growing complexity, and accurate, to capture the physically relevant features. To address this demand, we introduce a protein model that uses all-atom architecture to ensure the highest level of chemical detail while employing effective pair potentials to represent the effect of solvent to achieve the maximum speed. The effective potentials are derived for amino acid residues based on the condition that the solvent-free model matches the relevant pair-distribution functions observed in explicit solvent simulations. As a test, the model is applied to alanine polypeptides. For the chain with 10 amino acid residues, the model is found to reproduce properly the native state and its population. Small discrepancies are observed for other folding properties and can be attributed to the approximations inherent in the model. The transferability of the generated effective potentials is investigated in simulations of a longer peptide with 25 residues. A minimal set of potentials is identified that leads to qualitatively correct results in comparison with the explicit solvent simulations. Further tests, conducted for multiple peptide chains, show that the transferable model correctly reproduces the experimentally observed tendency of polyalanines to aggregate into β-sheets more strongly with the growing length of the peptide chain. Taken together, the reported results suggest that the proposed model could be used to succesfully simulate folding and aggregation of small peptides in atomic detail. Further tests are needed to assess the strengths and limitations of the model more thoroughly.

Biochemical Changes in Tissues during Infectious Illness: Metabolic Consequences of Interactions between Infectious Illness and Forced Exercise.

DTIC Science & Technology

1980-09-01

with the inoculation of S. typhimurium at 24 hrs post infection. Fasting ketosis was dramatically altered by the inoculation. Both plasma...as were both fasting and exercise associated ketosis . During fasting plasma Phydroxybutyrate increased 20-fold in the controls but only 13-fold in

Vocalization Subsystem Responses to a Temporarily Induced Unilateral Vocal Fold Paralysis

ERIC Educational Resources Information Center

Croake, Daniel J.; Andreatta, Richard D.; Stemple, Joseph C.

2018-01-01

Purpose: The purpose of this study is to quantify the interactions of the 3 vocalization subsystems of respiration, phonation, and resonance before, during, and after a perturbation to the larynx (temporarily induced unilateral vocal fold paralysis) in 10 vocally healthy participants. Using dynamic systems theory as a guide, we hypothesized that…

Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components

PubMed Central

Moparthi, Satish Babu; Carlsson, Uno; Vincentelli, Renaud; Jonsson, Bengt-Harald; Hammarström, Per; Wenger, Jérôme

2016-01-01

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of β-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes. PMID:27328749

Do Viruses Exchange Genes across Superkingdoms of Life?

PubMed

Malik, Shahana S; Azem-E-Zahra, Syeda; Kim, Kyung Mo; Caetano-Anollés, Gustavo; Nasir, Arshan

2017-01-01

Viruses can be classified into archaeoviruses, bacterioviruses, and eukaryoviruses according to the taxonomy of the infected host. The host-constrained perception of viruses implies preference of genetic exchange between viruses and cellular organisms of their host superkingdoms and viral origins from host cells either via escape or reduction. However, viruses frequently establish non-lytic interactions with organisms and endogenize into the genomes of bacterial endosymbionts that reside in eukaryotic cells. Such interactions create opportunities for genetic exchange between viruses and organisms of non-host superkingdoms. Here, we take an atypical approach to revisit virus-cell interactions by first identifying protein fold structures in the proteomes of archaeoviruses, bacterioviruses, and eukaryoviruses and second by tracing their spread in the proteomes of superkingdoms Archaea, Bacteria, and Eukarya. The exercise quantified protein structural homologies between viruses and organisms of their host and non-host superkingdoms and revealed likely candidates for virus-to-cell and cell-to-virus gene transfers. Unexpected lifestyle-driven genetic affiliations between bacterioviruses and Eukarya and eukaryoviruses and Bacteria were also predicted in addition to a large cohort of protein folds that were universally shared by viral and cellular proteomes and virus-specific protein folds not detected in cellular proteomes. These protein folds provide unique insights into viral origins and evolution that are generally difficult to recover with traditional sequence alignment-dependent evolutionary analyses owing to the fast mutation rates of viral gene sequences.

Role of the tail in the regulated state of myosin 2

PubMed Central

Jung, HyunSuk; Billington, Neil; Thirumurugan, Kavitha; Salzameda, Bridget; Cremo, Christine R.; Chalovich, Joseph M.; Chantler, Peter D.; Knight, Peter J.

2013-01-01

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together and the long tail is folded into three closely-packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. Using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Non-specific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation, and stabilises it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that folding of the tail is stabilised by ionic interactions between the positively-charged N-terminal sequence of the RLC and a negatively-charged region near the start of tail segment 3, and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilising the compact monomer conformation. PMID:21419133

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

PubMed Central

Paslawski, Wojciech; Lillelund, Ove K.; Kristensen, Julie Veje; Schafer, Nicholas P.; Baker, Rosanna P.; Urban, Sinisa; Otzen, Daniel E.

2015-01-01

Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated. PMID:26056273

Dispersion Interactions between Urea and Nucleobases Contribute to the Destabilization of RNA by Urea in Aqueous Solution

PubMed Central

Kasavajhala, Koushik; Bikkina, Swetha; Patil, Indrajit; MacKerell, Alexander D.; Priyakumar, U. Deva

2015-01-01

Urea has long been used to investigate protein folding and, more recently, RNA folding. Studies have proposed that urea denatures RNA by participating in stacking interactions and hydrogen bonds with nucleic acid bases. In this study, the ability of urea to form unconventional stacking interactions with RNA bases is investigated using ab initio calculations (RI-MP2 and CCSD(T) methods with the aug-cc-pVDZ basis set). A total of 29 stable nucleobase-urea stacked complexes are identified in which the intermolecular interaction energies (up to −14 kcal/mol) are dominated by dispersion effects. Natural bond orbital (NBO) and atoms in molecules (AIM) calculations further confirm strong interactions between urea and nucleobases. Calculations on model systems with multiple urea and water molecules interacting with a guanine base lead to a hypothesis that urea molecules along with water are able to form cage-like structures capable of trapping nucleic acid bases in extrahelical states by forming both hydrogen bonded and dispersion interactions, thereby contributing to the unfolding of RNA in the presence of urea in aqueous solution. PMID:25668757

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

PubMed Central

Joseph, Prem Raj B.; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M.; Garofalo, Roberto P.; Rajarathnam, Krishna

2013-01-01

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. PMID:24048001

Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad

PubMed Central

Cheong, Vee Vee; Heddi, Brahim; Lech, Christopher Jacques; Phan, Anh Tuân

2015-01-01

G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions. PMID:26400177

Development of liposomal and microemulsion formulations for transdermal delivery of clonazepam: effect of randomly methylated β-cyclodextrin.

PubMed

Mura, Paola; Bragagni, Marco; Mennini, Natascia; Cirri, Marzia; Maestrelli, Francesca

2014-11-20

Transdermal administration of clonazepam, a poorly water-soluble benzodiazepine, is an interesting strategy for overcoming the drawbacks of its oral administration. With this aim, two nano-carrier formulations, based on ultra-deformable liposomes and microemulsions, have been developed to favour clonazepam transdermal delivery. Considering the solubilizing power of methyl-βcyclodextrin (Me-βCD) toward clonazepam and its potential positive influence on transdermal drug delivery, the effect of its addition to these formulations was investigated. Artificial lipophilic membranes simulating the skin allowed a rapid evaluation of the drug permeation properties from the systems, compared with those from an aqueous drug suspension, with or without Me-βCD. The best formulations were further characterized by permeation through excised rabbit ear skin. All the formulations increased drug permeability, ranging from 2-fold (liposomes without Me-βCD), up to over 4-fold (microemulsions containing Me-βCD). The different formulations allowed for pointing out different possible permeation enhancing mechanisms of Me-βCD: increase in drug solubility and thermodynamic activity in the vehicle, when added to the drug aqueous suspension; interactions with the vesicle bilayer, in case of liposomal formulations; interactions with the skin membrane lipids, as evidenced in experiments with excised rabbit ear for microemulsions containing Me-βCD, that were then selected for further in vivo studies. Copyright © 2014. Published by Elsevier B.V.

Directed evolution of PDZ variants to generate high-affinity detection reagents.

PubMed

Ferrer, Marc; Maiolo, Jim; Kratz, Patricia; Jackowski, Jessica L; Murphy, Dennis J; Delagrave, Simon; Inglese, James

2005-04-01

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

Exploring the folding free energy landscape of insulin using bias exchange metadynamics.

PubMed

Todorova, Nevena; Marinelli, Fabrizio; Piana, Stefano; Yarovsky, Irene

2009-03-19

The bias exchange metadynamics (BE-META) technique was applied to investigate the folding mechanism of insulin, one of the most studied and biologically important proteins. The BE-META simulations were performed starting from an extended conformation of chain B of insulin, using only eight replicas and seven reaction coordinates. The folded state, together with the intermediate states along the folding pathway were identified and their free energy was determined. Three main basins were found separated from one another by a large free energy barrier. The characteristic native fold of chain B was observed in one basin, while the other two most populated basins contained "molten-globule" conformations stabilized by electrostatic and hydrophobic interactions, respectively. Transitions between the three basins occur on the microsecond time scale. The implications and relevance of this finding to the folding mechanisms of insulin were investigated.

A Particle Swarm Optimization-Based Approach with Local Search for Predicting Protein Folding.

PubMed

Yang, Cheng-Hong; Lin, Yu-Shiun; Chuang, Li-Yeh; Chang, Hsueh-Wei

2017-10-01

The hydrophobic-polar (HP) model is commonly used for predicting protein folding structures and hydrophobic interactions. This study developed a particle swarm optimization (PSO)-based algorithm combined with local search algorithms; specifically, the high exploration PSO (HEPSO) algorithm (which can execute global search processes) was combined with three local search algorithms (hill-climbing algorithm, greedy algorithm, and Tabu table), yielding the proposed HE-L-PSO algorithm. By using 20 known protein structures, we evaluated the performance of the HE-L-PSO algorithm in predicting protein folding in the HP model. The proposed HE-L-PSO algorithm exhibited favorable performance in predicting both short and long amino acid sequences with high reproducibility and stability, compared with seven reported algorithms. The HE-L-PSO algorithm yielded optimal solutions for all predicted protein folding structures. All HE-L-PSO-predicted protein folding structures possessed a hydrophobic core that is similar to normal protein folding.

Effects of adrenolytic mitotane on drug elimination pathways assessed in vitro.

PubMed

Theile, Dirk; Haefeli, Walter Emil; Weiss, Johanna

2015-08-01

Mitotane (1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane, o,p'-DDD) represents one of the most active drugs for the treatment of adrenocortical carcinoma. Its metabolites 1,1-(o,p'-dichlorodiphenyl) acetic acid (=o,p'-DDA) and 1,1-(o,p'-dichlorodiphenyl)-2,2 dichloroethene (=o,p'-DDE) partly contribute to its pharmacological effects. Because mitotane has a narrow therapeutic index and causes pharmacokinetic drug-drug interactions, knowledge about these compounds' effects on drug metabolizing and transporting proteins is crucial. Using quantitative real-time polymerase chain reaction, our study confirmed the strong inducing effects of o,p'-DDD on mRNA expression of cytochrome P450 3A4 (CYP3A4, 30-fold) and demonstrated that other enzymes and transporters are also induced (e.g., CYP1A2, 8.4-fold; ABCG2 (encoding breast resistance cancer protein, BCRP), 4.2-fold; ABCB1 (encoding P-glycoprotein, P-gp) 3.4-fold). P-gp induction was confirmed at the protein level. o,p'-DDE revealed a similar induction profile, however, with less potency and o,p'-DDA had only minor effects. Reporter gene assays clearly confirmed o,p'-DDD to be a PXR activator and for the first time demonstrated that o,p'-DDE and o,p'-DDA also activate PXR albeit with lower potency. Using isolated, recombinant CYP enzymes, o,p'-DDD and o,p'-DDE were shown to strongly inhibit CYP2C19 (IC50 = 0.05 and 0.09 µM). o,p'-DDA exhibited only minor inhibitory effects. In addition, o,p'-DDD, o,p'-DDE, and o,p'-DDA are demonstrated to be neither substrates nor inhibitors of BCRP or P-gp function. In summary, o,p'-DDD and o,p'-DDE might be potential perpetrators in pharmacokinetic drug-drug interactions through induction of drug-metabolizing enzymes or drug transporters and by potent inhibition of CYP2C19. In tumors over-expressing BCRP or P-gp, o,p'-DDD and its metabolites should retain their efficacy due to a lack of substrate characteristics.

A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.

PubMed

Ito, Shinya; Ogawa, Koji; Takeuchi, Koh; Takagi, Motoki; Yoshida, Masahito; Hirokawa, Takatsugu; Hirayama, Shoshiro; Shin-Ya, Kazuo; Shimada, Ichio; Doi, Takayuki; Goshima, Naoki; Natsume, Tohru; Nagata, Kazuhiro

2017-12-08

Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of urea and TMAO on the interaction between extended non-polar peptides

NASA Astrophysics Data System (ADS)

Su, Zhaoqian; Dias, Cristiano

Urea and trimethylamine n-oxide (TMAO) are small molecules known to destabilize and stabilize, respectively, the structure of proteins when added to aqueous solution. To unravel the molecular mechanisms of these cosolvents on protein structure we perform explicit all-atom molecular dynamics simulations of extended poly-alanine and polyleucine dimers. We use an umbrella sampling protocol to compute the potential of mean force (PMF) of dimers at different concentrations of urea and TMAO. We find that the large non-polar side chain of leucine is affected by urea whereas backbone atoms and alanine's side chain are not. Urea is found to occupy positions between leucine's side chains that are not accessible to water. This accounts for extra Lennard-Jones bonds between urea and side chains that favors the unfolded state. These bonds compete with urea-solvent interactions that favor the folded state. The sum of these two energetic terms provide the enthalpic driving force for unfolding. We show here that this enthalpy correlate with the potential of mean force of poly-leucine dimers. Moreover, the framework developed here is general and may be used to provide insights into effects of other small molecules on protein interactions. The effect of the TMAO will be in the presentation. Department of Physics, University Heights, Newark, New Jersey, 07102-1982.

Effect of solvation-related interaction on the low-temperature dynamics of proteins

NASA Astrophysics Data System (ADS)

Zuo, Guanghong; Wang, Jun; Qin, Meng; Xue, Bin; Wang, Wei

2010-03-01

The effect of solvation-related interaction on the low-temperature dynamics of proteins is studied by taking into account the desolvation barriers in the interactions of native contacts. It is found out that about the folding transition temperature, the protein folds in a cooperative manner, and the water molecules are expelled from the hydrophobic core at the final stage in the folding process. At low temperature, however, the protein would generally be trapped in many metastable conformations with some water molecules frozen inside the protein. The desolvation takes an important role in these processes. The number of frozen water molecules and that of frozen states of proteins are further analyzed with the methods based on principal component analysis (PCA) and the clustering of conformations. It is found out that both the numbers of frozen water molecules and the frozen states of the protein increase quickly below a certain temperature. Especially, the number of frozen states of the protein increases exponentially following the decrease in the temperature, which resembles the basic features of glassy dynamics. Interestingly, it is observed that the freezing of water molecules and that of protein conformations happen at almost the same temperature. This suggests that the solvation-related interaction performs an important role for the low-temperature dynamics of the model protein.

Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

PubMed Central

Borgia, Alessandro; Wensley, Beth G.; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B.; Hoffmann, Armin; Pfeil, Shawn H.; Lipman, Everett A.; Clarke, Jane; Schuler, Benjamin

2012-01-01

Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes. PMID:23149740

Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy.

PubMed

Borgia, Alessandro; Wensley, Beth G; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B; Hoffmann, Armin; Pfeil, Shawn H; Lipman, Everett A; Clarke, Jane; Schuler, Benjamin

2012-01-01

Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes.

Gas and Shadow Swing

NASA Astrophysics Data System (ADS)

Tsai, Chi-Hung; Lai, Mei-Yi; Liu, Che-Wei; Huang, Shiang-Yin; Lin, Che-Yu; Yeh, Jeng-Sheng

In our digital art, we design a folding fan as an interactive magic device. You can use it to play with gas around the world of illusions. Although gas could not be seen in our real world, we still want to interact with it in our illusions by the element of bubble shadows. Opening and swinging the folding fan can blow the bubble shadows away; closing and swinging it can break bubbles. If the magic fan touches the shadow of gas, the bubble shadows will explode and release colorful particles to surround you. Those actions are controlled and located by our circuits with Arduino board.

Soil solution interactions may limit Pb remediation using P ...

EPA Pesticide Factsheets

Lead (Pb) contaminated soils are a potential exposure hazard to the public. Amending soils with phosphorus (P) may reduce Pb soil hazards. Soil from Cleveland, OH containing 726 ± 14 mg Pb kg-1 was amended in a laboratory study with bone meal and triple super phosphate (TSP) at 5:1 P:Pb molar ratios. Soil was acidified, neturalized and re-acidified to encourage Pb phosphate formation. PRSTM-probes were used to evaluate changes in soil solution chemistry. Soil acidification did not decrease in vitro bioaccessible (IVBA) Pb using either a pH 1.5, 0.4 M glycine solution or a pH 2.5 solution with organic acids. PRSTM-probe data found soluble Pb increased 10-fold in acidic conditions compared to circumnetural pH conditions. In acidic conditions (p = 3-4), TSP treated soils increased detected P 10-fold over untreated soils. Bone meal application did not increase PRSTM-probe detected P, indicating there may have been insufficient P to react with Pb. X-ray absorption spectroscopy suggested a 10% increase in pyromorphite formation for the TSP treated soil only. Treatments increased soil electrical conductivity above 16 mS cm-1, potentially causing a new salinity hazard. This study used a novel approach by combining the human ingestion endpoint, PRSTM-probes, and X-ray absorption spectroscopy to evaluate treatment efficacy. PRSTM-probe data indicated potentially excess Ca relative to P across incubation steps that could have competed with Pb for soluble P. Mor

Sulforaphane Potentiates the Efficacy of 17-Allylamino 17-Demethoxygeldanamycin Against Pancreatic Cancer Through Enhanced Abrogation of Hsp90 Chaperone Function

PubMed Central

Li, Yanyan; Zhang, Tao; Schwartz, Steven J.; Sun, Duxin

2013-01-01

Heat shock protein 90 (Hsp90), an essential molecular chaperone that regulates the stability of a wide range of oncogenic proteins, is a promising target for cancer therapeutics. We investigated the combination efficacy and potential mechanisms of sulforaphane, a dietary component from broccoli and broccoli sprouts, and 17-allylamino 17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, in pancreatic cancer. MTS assay demonstrated that sulforaphane sensitized pancreatic cancer cells to 17-AAG in vitro. Caspase-3 was activated to 6.4-fold in response to simultaneous treatment with sulforaphane and 17-AAG, whereas 17-AAG alone induced caspase-3 activity to 2-fold compared to control. ATP binding assay and coimmunoprecipitation revealed that sulforaphane disrupted Hsp90-p50Cdc37 interaction, whereas 17-AAG inhibited ATP binding to Hsp90. Concomitant use of sulforaphane and 17-AAG synergistically downregulated Hsp90 client proteins in Mia Paca-2 cells. Co-administration of sulforaphane and 17-AAG in pancreatic cancer xenograft model led to more than 70% inhibition of the tumor growth, whereas 17-AAG alone only suppressed the tumor growth by 50%. Our data suggest that sulforaphane potentiates the efficacy of 17-AAG against pancreatic cancer through enhanced abrogation of Hsp90 function. These findings provide a rationale for further evaluation of broccoli/broccoli sprout preparations combined with 17-AAG for better efficacy and lower dose-limiting toxicity in pancreatic cancer. PMID:21875325

Thermodynamic properties of an extremely rapid protein folding reaction.

PubMed

Schindler, T; Schmid, F X

1996-12-24

The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to avoid misfolding prior to the rate-limiting step, and a native-like activated state reduces the risk of non-productive side reactions during the final steps to the native state.

A thermodynamic definition of protein domains.

PubMed

Porter, Lauren L; Rose, George D

2012-06-12

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

Complete fold annotation of the human proteome using a novel structural feature space.

PubMed

Middleton, Sarah A; Illuminati, Joseph; Kim, Junhyong

2017-04-13

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.

Complete fold annotation of the human proteome using a novel structural feature space

PubMed Central

Middleton, Sarah A.; Illuminati, Joseph; Kim, Junhyong

2017-01-01

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families. PMID:28406174

Capsaicin pretreatment enhanced the bioavailability of fexofenadine in rats by P-glycoprotein modulation: in vitro, in situ and in vivo evaluation.

PubMed

Bedada, Satish Kumar; Appani, Ramgopal; Boga, Praveen Kumar

2017-06-01

Capsaicin is the main pungent principle present in chili peppers has been found to possess P-glycoprotein (P-gp) inhibition activity in vitro, which may have the potential to modulate bioavailability of P-gp substrates. Therefore, purpose of this study was to evaluate the effect of capsaicin on intestinal absorption and bioavailability of fexofenadine, a P-gp substrate in rats. The mechanistic evaluation was determined by non-everted sac and intestinal perfusion studies to explore the intestinal absorption of fexofenadine. These results were confirmed by an in vivo pharmacokinetic study of oral administered fexofenadine in rats. The intestinal transport and apparent permeability (P app ) of fexofenadine were increased significantly by 2.8 and 2.6 fold, respectively, in ileum of capsaicin treated rats when compared to control group. Similarly, absorption rate constant (K a ), fraction absorbed (F ab ) and effective permeability (P eff ) of fexofenadine were increased significantly by 2.8, 2.9 and 3.4 fold, respectively, in ileum of rats pretreated with capsaicin when compared to control group. In addition, maximum plasma concentration (C max ) and area under the concentration-time curve (AUC) were increased significantly by 2.3 and 2.4 fold, respectively, in rats pretreated with capsaicin as compared to control group. Furthermore, obtained results in rats pretreated with capsaicin were comparable to verapamil (positive control) treated rats. Capsaicin pretreatment significantly enhanced the intestinal absorption and bioavailability of fexofenadine in rats likely by inhibition of P-gp mediated cellular efflux, suggesting that the combined use of capsaicin with P-gp substrates may require close monitoring for potential drug interactions.

The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota

PubMed Central

Cox, Laura M.; Cho, Ilseung; Young, Scott A.; Anderson, W. H. Kerr; Waters, Bartholomew J.; Hung, Shao-Ching; Gao, Zhan; Mahana, Douglas; Bihan, Monika; Alekseyenko, Alexander V.; Methé, Barbara A.; Blaser, Martin J.

2013-01-01

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.—Cox, L. M., Cho, I., Young, S. A., Kerr Anderson, W. H., Waters, B. J., Hung, S.-C., Gao, Z., Mahana, D., Bihan, M., Alekseyenko, A. V., Methé, B. A., Blaser, M. J. The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota. PMID:23154883

Domains of Pyrococcus furiosus L-asparaginase fold sequentially and assemble through strong intersubunit associative forces.

PubMed

Garg, Dushyant K; Tomar, Rachana; Dhoke, Reema R; Srivastava, Ankit; Kundu, Bishwajit

2015-05-01

Here, we report the folding and assembly of a Pyrococcus furiosus-derived protein, L-asparaginase (PfA). PfA functions as a homodimer, with each monomer made of distinct N- and C-terminal domains. The purified individual domains as well as single Trp mutant of each domain were subjected to chemical denaturation/renaturation and probed by combination of spectroscopic, chromatographic, quenching and scattering techniques. We found that the N-domain acts like a folding scaffold and assists the folding of remaining polypeptide. The domains displayed sequential folding with the N-domain having higher thermodynamic stability. We report that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network. Our results proved that folding cooperativity in a thermophilic, multisubunit protein is dictated by concomitant folding and association of constituent domains directly into a native quaternary structure. This report gives an account of the factors responsible for folding and stability of a therapeutically and industrially important protein.

A semi-analytical description of protein folding that incorporates detailed geometrical information

PubMed Central

Suzuki, Yoko; Noel, Jeffrey K.; Onuchic, José N.

2011-01-01

Much has been done to study the interplay between geometric and energetic effects on the protein folding energy landscape. Numerical techniques such as molecular dynamics simulations are able to maintain a precise geometrical representation of the protein. Analytical approaches, however, often focus on the energetic aspects of folding, including geometrical information only in an average way. Here, we investigate a semi-analytical expression of folding that explicitly includes geometrical effects. We consider a Hamiltonian corresponding to a Gaussian filament with structure-based interactions. The model captures local features of protein folding often averaged over by mean-field theories, for example, loop contact formation and excluded volume. We explore the thermodynamics and folding mechanisms of beta-hairpin and alpha-helical structures as functions of temperature and Q, the fraction of native contacts formed. Excluded volume is shown to be an important component of a protein Hamiltonian, since it both dominates the cooperativity of the folding transition and alters folding mechanisms. Understanding geometrical effects in analytical formulae will help illuminate the consequences of the approximations required for the study of larger proteins. PMID:21721664

Functional Tat transport of unstructured, small, hydrophilic proteins.

PubMed

Richter, Silke; Lindenstrauss, Ute; Lücke, Christian; Bayliss, Richard; Brüser, Thomas

2007-11-16

The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.

Nature of Driving Force for Protein Folding-- A Result From Analyzing the Statistical Potential

NASA Astrophysics Data System (ADS)

Li, Hao; Tang, Chao; Wingreen, Ned S.

1998-03-01

In a statistical approach to protein structure analysis, Miyazawa and Jernigan (MJ) derived a 20× 20 matrix of inter-residue contact energies between different types of amino acids. Using the method of eigenvalue decomposition, we find that the MJ matrix can be accurately reconstructed from its first two principal component vectors as M_ij=C_0+C_1(q_i+q_j)+C2 qi q_j, with constant C's, and 20 q values associated with the 20 amino acids. This regularity is due to hydrophobic interactions and a force of demixing, the latter obeying Hildebrand's solubility theory of simple liquids.

Preliminary chaotic model of snapover on high voltage solar cells

NASA Technical Reports Server (NTRS)

Mackey, Willie R.

1995-01-01

High voltage power systems in space will interact with the space plasma in a variety of ways. One of these, snapover, is characterized by sudden enlargement of the current collection area across normally insulating surfaces generating enhanced electron current collection. Power drain on solar array power systems results from this enhanced current collection. Optical observations of the snapover phenomena in the laboratory indicates a functional relation between glow area and bia potential as a consequence of the fold/cusp bifurcation in chaos theory. Successful characterizations of snapover as a chaotic phenomena may provide a means of snapover prevention and control through chaotic synchronization.

High-throughput SRCD using multi-well plates and its applications

NASA Astrophysics Data System (ADS)

Hussain, Rohanah; Jávorfi, Tamás; Rudd, Timothy R.; Siligardi, Giuliano

2016-12-01

The sample compartment for high-throughput synchrotron radiation circular dichroism (HT-SRCD) has been developed to satisfy an increased demand of protein characterisation in terms of folding and binding interaction properties not only in the traditional field of structural biology but also in the growing research area of material science with the potential to save time by 80%. As the understanding of protein behaviour in different solvent environments has increased dramatically the development of novel functions such as recombinant proteins modified to have different functions from harvesting solar energy to metabolonics for cleaning heavy and metal and organic molecule pollutions, there is a need to characterise speedily these system.

The importance of hyaluronic acid in vocal fold biomechanics.

PubMed

Chan, R W; Gray, S D; Titze, I R

2001-06-01

This study examined the influence of hyaluronic acid (HA) on the biomechanical properties of the human vocal fold cover (the superficial layer of the lamina propria). Vocal fold tissues were freshly excised from 5 adult male cadavers and were treated with bovine testicular hyaluronidase to selectively remove HA from the lamina propria extracellular matrix (ECM). Linear viscoelastic shear properties (elastic shear modulus and dynamic viscosity) of the tissue samples before and after enzymatic treatment were quantified as a function of frequency (0.01 to 15 Hz) by a parallel-plate rotational rheometer at 37 degrees C. On removing HA from the vocal fold ECM, the elastic shear modulus (G' ) or stiffness of the vocal fold cover decreased by an average of around 35%, while the dynamic viscosity (eta') increased by 70% at higher frequencies (>1 Hz). The results suggested that HA plays an important role in determining the biomechanical properties of the vocal fold cover. As a highly hydrated glycosaminoglycan in the vocal fold ECM, it likely contributes to the maintenance of an optimal tissue viscosity that may facilitate phonation, and an optimal tissue stiffness that may be important for vocal fundamental frequency control. HA has been proposed as a potential bioimplant for the surgical repair of vocal fold ECM defects (eg, vocal fold scarring and sulcus vocalis). Our results suggested that such clinical use may be potentially optimal for voice production from a biomechanical perspective.

Investigating the Mechanisms and Potential of Silk Fiber Metallization

DTIC Science & Technology

2013-09-30

in an ALD process, when using the modified metal infiltration process as outlined by Lee et al., the titanium isopropoxide (TIP) precursor...these fibers exhibited >2-fold increase in strain to breakage, and >4.5-fold increase in strength when infiltrated with zinc, titanium , or aluminum...fibers exhibited >2-fold increase in strain to breakage, and >4.5-fold increase in strength when infiltrated with zinc, titanium , or aluminum

A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction.

PubMed

Li, Ying; Xu, Fei; Liu, Chao; Xu, Youzhi; Feng, Xiaojun; Liu, Bi-Feng

2013-08-21

Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

MRP2 mediated drug-drug interaction: indomethacin increases sulfasalazine absorption in the small intestine, potentially decreasing its colonic targeting.

PubMed

Dahan, Arik; Amidon, Gordon L

2010-02-15

We have recently shown that efflux transport, mediated by multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP), is responsible for sulfasalazine low-permeability in the small intestine, thereby enabling its colonic targeting and therapeutic action. The purpose of the present study was to evaluate the potential pharmacokinetic interaction between indomethacin and sulfasalazine, in the mechanism of efflux transporter competition. The concentration-dependent effects of indomethacin on sulfasalazine intestinal epithelial transport were investigated across Caco-2 cell monolayers, in both apical to basolateral (AP-BL) and BL-AP directions. The interaction was then investigated in the in situ single-pass rat jejunal perfusion model. Sulfasalazine displayed 30-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. Indomethacin significantly increased AP-BL and decreased BL-AP sulfasalazine Caco-2 transport, in a concentration-dependent manner, with IC(50) values of 75 and 196 microM respectively. In the rat model, higher sulfasalazine concentrations resulted in higher intestinal permeability, consistent with saturation of efflux transporter. Without indomethacin, sulfasalazine demonstrated low rat jejunal permeability (vs. metoprolol). Indomethacin significantly increased sulfasalazine P(eff), effectively shifting it from BCS (biopharmaceutics classification system) Class IV to II. In conclusion, the data indicate that concomitant intake of indomethacin and sulfasalazine may lead to increased absorption of sulfasalazine in the small intestine, thereby reducing its colonic concentration and potentially altering its therapeutic effect. Copyright 2009 Elsevier B.V. All rights reserved.

Accelerated molecular dynamics simulations of protein folding.

PubMed

Miao, Yinglong; Feixas, Ferran; Eun, Changsun; McCammon, J Andrew

2015-07-30

Folding of four fast-folding proteins, including chignolin, Trp-cage, villin headpiece and WW domain, was simulated via accelerated molecular dynamics (aMD). In comparison with hundred-of-microsecond timescale conventional molecular dynamics (cMD) simulations performed on the Anton supercomputer, aMD captured complete folding of the four proteins in significantly shorter simulation time. The folded protein conformations were found within 0.2-2.1 Å of the native NMR or X-ray crystal structures. Free energy profiles calculated through improved reweighting of the aMD simulations using cumulant expansion to the second-order are in good agreement with those obtained from cMD simulations. This allows us to identify distinct conformational states (e.g., unfolded and intermediate) other than the native structure and the protein folding energy barriers. Detailed analysis of protein secondary structures and local key residue interactions provided important insights into the protein folding pathways. Furthermore, the selections of force fields and aMD simulation parameters are discussed in detail. Our work shows usefulness and accuracy of aMD in studying protein folding, providing basic references in using aMD in future protein-folding studies. © 2015 Wiley Periodicals, Inc.

Folding of the four-helix bundle FF domain from a compact on-pathway intermediate state is governed predominantly by water motion.

PubMed

Sekhar, Ashok; Vallurupalli, Pramodh; Kay, Lewis E

2012-11-20

Friction plays a critical role in protein folding. Frictional forces originating from random solvent and protein fluctuations both retard motion along the folding pathway and activate protein molecules to cross free energy barriers. Studies of friction thus may provide insights into the driving forces underlying protein conformational dynamics. However, the molecular origin of friction in protein folding remains poorly understood because, with the exception of the native conformer, there generally is little detailed structural information on the other states participating in the folding process. Here, we study the folding of the four-helix bundle FF domain that proceeds via a transiently formed, sparsely populated compact on-pathway folding intermediate whose structure was elucidated previously. Because the intermediate is stabilized by both native and nonnative interactions, friction in the folding transition between intermediate and folded states is expected to arise from intrachain reorganization in the protein. However, the viscosity dependencies of rates of folding from or unfolding to the intermediate, as established by relaxation dispersion NMR spectroscopy, clearly indicate that contributions from internal friction are small relative to those from solvent, so solvent frictional forces drive the folding process. Our results emphasize the importance of solvent dynamics in mediating the interconversion between protein configurations, even those that are highly compact, and in equilibrium folding/unfolding fluctuations in general.

Contribution of Charged Groups to the Enthalpic Stabilization of the Folded States of Globular Proteins

PubMed Central

Dadarlat, Voichita M.; Post, Carol Beth

2016-01-01

In this paper we use the results from all atom MD simulations of proteins and peptides to assess individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the “unfolded state”) and charged amino acids in globular proteins (the “folded state”). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO−) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while NH3+ groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously. PMID:18303881

Maximizing RNA folding rates: a balancing act.

PubMed Central

Thirumalai, D; Woodson, S A

2000-01-01

Large ribozymes typically require very long times to refold into their active conformation in vitro, because the RNA is easily trapped in metastable misfolded structures. Theoretical models show that the probability of misfolding is reduced when local and long-range interactions in the RNA are balanced. Using the folding kinetics of the Tetrahymena ribozyme as an example, we propose that folding rates are maximized when the free energies of forming independent domains are similar to each other. A prediction is that the folding pathway of the ribozyme can be reversed by inverting the relative stability of the tertiary domains. This result suggests strategies for optimizing ribozyme sequences for therapeutics and structural studies. PMID:10864039

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

PubMed Central

Lundin, Victor F.; Stirling, Peter C.; Gomez-Reino, Juan; Mwenifumbo, Jill C.; Obst, Jennifer M.; Valpuesta, José M.; Leroux, Michel R.

2004-01-01

Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides. PMID:15070724

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Individual and combined effects of maternal anemia and prenatal infection on risk for schizophrenia in offspring.

PubMed

Nielsen, Philip R; Meyer, Urs; Mortensen, Preben B

2016-04-01

Maternal iron deficiency and infection during pregnancy have individually been associated with increased risk of schizophrenia in the offspring, but possible interactions between the two remain unidentified thus far. Therefore, we determined the individual and combined effects of maternal infection during pregnancy and prepartum anemia on schizophrenia risk in the offspring. We conducted a population-based study with individual record linkage of the Danish Civil Registration System, the Danish Hospital Register, and the Central Danish Psychiatric Register. In a cohort of Danish singleton births 1,403,183 born between 1977 and 2002, 6729 developed schizophrenia between 1987 and 2012. Cohort members were considered as having a maternal history of anemia if the mother had received a diagnosis of anemia at any time during the pregnancy. Maternal infection was defined based on infections requiring hospital admission during pregnancy. Maternal anemia and infection were both associated with increased risk of schizophrenia in unadjusted analyses (1.45-fold increase for anemia, 95% CI: 1.14-1.82; 1.32-fold increase for infection, 95% CI: 1.17-1.48). The effect of maternal infection remained significant (1.16-fold increase, 95% CI: 1.03-1.31) after adjustment for possible confounding factors. Combined exposure to anemia and an infection increased the effect size to a 2.49-fold increased schizophrenia risk (95% CI: 1.29-4.27). The interaction analysis, however, failed to provide evidence for multiplicative interactions between the two factors. Our findings indicate that maternal anemia and infection have additive but not interactive effects, and therefore, they may represent two independent risk factors of schizophrenia. Copyright © 2016 Elsevier B.V. All rights reserved.

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum.

PubMed

Ishikawa, Yoshihiro; Holden, Paul; Bächinger, Hans Peter

2017-10-20

Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/ SERPINH1 ) and 65-kDa FK506-binding protein (FKBP65/ FKBP10 ), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Middleton, Sarah A.; Illuminati, Joseph; Kim, Junhyong

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this methodmore » by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Finally, our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.« less

Isolation, Characterization and Lipid-Binding Properties of the Recalcitrant FtsA Division Protein from Escherichia coli

PubMed Central

Zorrilla, Silvia; Reija, Belén; Alfonso, Carlos; Mingorance, Jesús; Rivas, Germán; Jiménez, Mercedes

2012-01-01

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures. PMID:22761913

Vocal fold hemorrhage associated with coumadin therapy in an opera singer.

PubMed

Neely, J L; Rosen, C

2000-06-01

Vocal fold hemorrhage can represent a disastrous and potentially career ending injury to a singer or professional voice user. The risk factors of vocal fold hemorrhage, including laryngeal trauma, phonotrauma, aspirin and nonsteroidal antiinflammatories, and hormonal imbalances are well known. We present a case of an opera singer who developed recurrent vocal fold hemorrhage associated with coumadin anticoagulation therapy. This case highlights the importance of the risk of vocal fold hemorrhage to professional singers and professional voice users and offers an alternative to long-term coumadin therapy in this select population.

Impact of the CYP2C8 *3 polymorphism on the drug–drug interaction between gemfibrozil and pioglitazone

PubMed Central

Aquilante, Christina L; Kosmiski, Lisa A; Bourne, David W A; Bushman, Lane R; Daily, Elizabeth B; Hammond, Kyle P; Hopley, Charles W; Kadam, Rajendra S; Kanack, Alexander T; Kompella, Uday B; Le, Merry; Predhomme, Julie A; Rower, Joseph E; Sidhom, Maha S

2013-01-01

AIM The objective of this study was to determine the extent to which the CYP2C8*3 allele influences pharmacokinetic variability in the drug–drug interaction between gemfibrozil (CYP2C8 inhibitor) and pioglitazone (CYP2C8 substrate). METHODS In this randomized, two phase crossover study, 30 healthy Caucasian subjects were enrolled based on CYP2C8*3 genotype (n = 15, CYP2C8*1/*1; n = 15, CYP2C8*3 carriers). Subjects received a single 15 mg dose of pioglitazone or gemfibrozil 600 mg every 12 h for 4 days with a single 15 mg dose of pioglitazone administered on the morning of day 3. A 48 h pharmacokinetic study followed each pioglitazone dose and the study phases were separated by a 14 day washout period. RESULTS Gemfibrozil significantly increased mean pioglitazone AUC(0,∞) by 4.3-fold (P < 0.001) and there was interindividual variability in the magnitude of this interaction (range, 1.8- to 12.1-fold). When pioglitazone was administered alone, the mean AUC(0,∞) was 29.7% lower (P= 0.01) in CYP2C8*3 carriers compared with CYP2C8*1 homozygotes. The relative change in pioglitazone plasma exposure following gemfibrozil administration was significantly influenced by CYP2C8 genotype. Specifically, CYP2C8*3 carriers had a 5.2-fold mean increase in pioglitazone AUC(0,∞) compared with a 3.3-fold mean increase in CYP2C8*1 homozygotes (P= 0.02). CONCLUSION CYP2C8*3 is associated with decreased pioglitazone plasma exposure in vivo and significantly influences the pharmacokinetic magnitude of the gemfibrozil–pioglitazone drug-drug interaction. Additional studies are needed to evaluate the impact of CYP2C8 genetics on the pharmacokinetics of other CYP2C8-mediated drug–drug interactions. PMID:22625877

Impact of the CYP2C8 *3 polymorphism on the drug-drug interaction between gemfibrozil and pioglitazone.

PubMed

Aquilante, Christina L; Kosmiski, Lisa A; Bourne, David W A; Bushman, Lane R; Daily, Elizabeth B; Hammond, Kyle P; Hopley, Charles W; Kadam, Rajendra S; Kanack, Alexander T; Kompella, Uday B; Le, Merry; Predhomme, Julie A; Rower, Joseph E; Sidhom, Maha S

2013-01-01

The objective of this study was to determine the extent to which the CYP2C8*3 allele influences pharmacokinetic variability in the drug-drug interaction between gemfibrozil (CYP2C8 inhibitor) and pioglitazone (CYP2C8 substrate). In this randomized, two phase crossover study, 30 healthy Caucasian subjects were enrolled based on CYP2C8*3 genotype (n = 15, CYP2C8*1/*1; n = 15, CYP2C8*3 carriers). Subjects received a single 15 mg dose of pioglitazone or gemfibrozil 600 mg every 12 h for 4 days with a single 15 mg dose of pioglitazone administered on the morning of day 3. A 48 h pharmacokinetic study followed each pioglitazone dose and the study phases were separated by a 14 day washout period. Gemfibrozil significantly increased mean pioglitazone AUC(0,∞) by 4.3-fold (P < 0.001) and there was interindividual variability in the magnitude of this interaction (range, 1.8- to 12.1-fold). When pioglitazone was administered alone, the mean AUC(0,∞) was 29.7% lower (P = 0.01) in CYP2C8*3 carriers compared with CYP2C8*1 homozygotes. The relative change in pioglitazone plasma exposure following gemfibrozil administration was significantly influenced by CYP2C8 genotype. Specifically, CYP2C8*3 carriers had a 5.2-fold mean increase in pioglitazone AUC(0,∞) compared with a 3.3-fold mean increase in CYP2C8*1 homozygotes (P = 0.02). CYP2C8*3 is associated with decreased pioglitazone plasma exposure in vivo and significantly influences the pharmacokinetic magnitude of the gemfibrozil-pioglitazone drug-drug interaction. Additional studies are needed to evaluate the impact of CYP2C8 genetics on the pharmacokinetics of other CYP2C8-mediated drug-drug interactions. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins.

PubMed

Mathur, Puniti; Ramakumar, S; Chauhan, V S

2004-01-01

Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures. Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate.

PubMed

Zako, Tamotsu; Murase, Yosuke; Iizuka, Ryo; Yoshida, Takao; Kanzaki, Taro; Ide, Naoki; Maeda, Mizuo; Funatsu, Takashi; Yohda, Masafumi

2006-11-17

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin.

Structure of the Mtb CarD/RNAP β-lobes complex reveals the molecular basis of interaction and presents a distinct DNA-binding domain for Mtb CarD.

PubMed

Gulten, Gulcin; Sacchettini, James C

2013-10-08

CarD from Mycobacterium tuberculosis (Mtb) is an essential protein shown to be involved in stringent response through downregulation of rRNA and ribosomal protein genes. CarD interacts with the β-subunit of RNAP and this interaction is vital for Mtb's survival during the persistent infection state. We have determined the crystal structure of CarD in complex with the RNAP β-subunit β1 and β2 domains at 2.1 Å resolution. The structure reveals the molecular basis of CarD/RNAP interaction, providing a basis to further our understanding of RNAP regulation by CarD. The structural fold of the CarD N-terminal domain is conserved in RNAP interacting proteins such as TRCF-RID and CdnL, and displays similar interactions to the predicted homology model based on the TRCF/RNAP β1 structure. Interestingly, the structure of the C-terminal domain, which is required for complete CarD function in vivo, represents a distinct DNA-binding fold. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vivo pharmacokinetic interaction by ethanolic extract of Gymnema sylvestre with CYP2C9 (Tolbutamide), CYP3A4 (Amlodipine) and CYP1A2 (Phenacetin) in rats.

PubMed

Vaghela, Madhuri; Sahu, Niteshkumar; Kharkar, Prashant; Pandita, Nancy

2017-12-25

Gymnema sylvestre (GS) is a medicinal herb used for diabetes mellitus (DM). Herbs are gaining popularity as medicines in DM for its safety purpose. The aim of the present study was to evaluate in vivo pharmacokinetic (PK) interaction between allopathic drugs tolbutamide (TOLBU), amlodipine (AMLO), and phenacetin (PHENA) at low (L) and high (H) doses with ethanolic extract (EL) from GS. EL was extracted and subjected to TLC, total triterpenoid content (19.76 ± 0.02 W/W) and sterol content (0.1837 ± 0.0046 W/W) estimation followed by identification of phytoconstituents using HRLC-MS and GC-MS. PK interaction study with CYP2C9, CYP3A4 and CYP1A2 enzymes were assessed using TOLBU, AMLO and PHENA respectively to index cytochrome (CYP) mediated interaction in rats after concomitant administration of EL extract (400 mg/kg) from GS for 7 days. The rats were divided into four groups for each PK study where, group I and II were positive control for low and high dose of test drugs (CYP substrates) while group II and IV were orally administered EL. The PK study result of PHENA indicated that area under the plasma concentration-time curve (AUC 0-24 ) was significantly (P < 0.0001) increased by 1.4 (L) and 1.3-fold (H), plasma concentration (C max ) was significantly (P < 0.001) increased by 1.6 (L) and 1.4-fold (H). Whereas for TOLBU; clearance rate (CL) was significantly (P < 0.0001) decreased by 2.4 (L) and 2.3-fold (H), C max, was significantly (P < 0.001) decreased by 26.5% (L) and 50.4% (H) and AUC 0-24 was significantly (P < 0.0001) decreased by 59.8% (L) and 57.5% (H). Thus, EL is seen to be interacting with CYP1A2 by inhibiting its metabolic activity. HRLC-MS and GC-MS helped identify the presence of gymnemic acid (GA), triterpenoids and steroids in EL which could be the reason for PK interaction of CYP1A2 and CYP2C9. Also, in silico structure based site of metabolism study showed Fe accessibility and intrinsic activity for GA-IV, GA-VI, GA-VII and GA-X with CYP2C9. PK parameters of AMLO were not significantly affected by pre-treatment of EL. Thereby our findings indicate that co-administration of GS with drugs that are metabolized by CYP2C9 and CYP1A2 could lead to potential HDI. Copyright © 2017. Published by Elsevier B.V.

Folds on Europa: implications for crustal cycling and accommodation of extension.

PubMed

Prockter, L M; Pappalardo, R T

2000-08-11

Regional-scale undulations with associated small-scale secondary structures are inferred to be folds on Jupiter's moon Europa. Formation is consistent with stresses from tidal deformation, potentially triggering compressional instability of a region of locally high thermal gradient. Folds may compensate for extension elsewhere on Europa and then relax away over time.

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates

PubMed Central

Bhat, Shreyas; Hasenhuetl, Peter S.; Kasture, Ameya; El-Kasaby, Ali; Baumann, Michael H.; Blough, Bruce E.; Sucic, Sonja; Sandtner, Walter; Freissmuth, Michael

2017-01-01

Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to μm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants. PMID:28842491

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

PubMed Central

Dumont, Jennifer A.; Liu, Tongyao; Low, Susan C.; Zhang, Xin; Kamphaus, George; Sakorafas, Paul; Fraley, Cara; Drager, Douglas; Reidy, Thomas; McCue, Justin; Franck, Helen W. G.; Merricks, Elizabeth P.; Nichols, Timothy C.; Bitonti, Alan J.; Pierce, Glenn F.

2012-01-01

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation. PMID:22246033

Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyuhas, Ronit; Noy, Hava; Fishman, Sigal

2009-08-21

HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect onmore » 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.« less

On the orientation of the backbone dipoles in native folds

PubMed Central

Ripoll, Daniel R.; Vila, Jorge A.; Scheraga, Harold A.

2005-01-01

The role of electrostatic interactions in determining the native fold of proteins has been investigated by analyzing the alignment of peptide bond dipole moments with the local electrostatic field generated by the rest of the molecule with and without solvent effects. This alignment was calculated for a set of 112 native proteins by using charges from a gas phase potential. Most of the peptide dipoles in this set of proteins are on average aligned with the electrostatic field. The dipole moments associated with α-helical conformations show the best alignment with the electrostatic field, followed by residues in β-strand conformations. The dipole moments associated with other secondary structure elements are on average better aligned than in randomly generated conformations. The alignment of a dipole with the local electrostatic field depends on both the topology of the native fold and the charge distribution assumed for all of the residues. The influences of (i) solvent effects, (ii) different sets of charges, and (iii) the charge distribution assumed for the whole molecule were examined with a subset of 22 proteins each of which contains <30 ionizable groups. The results show that alternative charge distribution models lead to significant differences among the associated electrostatic fields, whereas the electrostatic field is less sensitive to the particular set of the adopted charges themselves (empirical conformational energy program for peptides or parameters for solvation energy). PMID:15894608

Vibrational dynamics of vocal folds using nonlinear normal modes.

PubMed

Pinheiro, Alan P; Kerschen, Gaëtan

2013-08-01

Many previous works involving physical models, excised and in vivo larynges have pointed out nonlinear vibration in vocal folds during voice production. Moreover, theoretical studies involving mechanical modeling of these folds have tried to gain a profound understanding of the observed nonlinear phenomena. In this context, the present work uses the nonlinear normal mode theory to investigate the nonlinear modal behavior of 16 subjects using a two-mass mechanical modeling of the vocal folds. The free response of the conservative system at different energy levels is considered to assess the impact of the structural nonlinearity of the vocal fold tissues. The results show very interesting and complex nonlinear phenomena including frequency-energy dependence, subharmonic regimes and, in some cases, modal interactions, entrainment and bifurcations. Copyright © 2012 IPEM. Published by Elsevier Ltd. All rights reserved.

Predicting loop–helix tertiary structural contacts in RNA pseudoknots

PubMed Central

Cao, Song; Giedroc, David P.; Chen, Shi-Jie

2010-01-01

Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop–stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop–stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop–stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory–experimental comparisons. These comparisons reveal a contact enthalpy (ΔH) of −14 kcal/mol and a contact entropy (ΔS) of −38 cal/mol/K for a protonated C+•(G–C) base triple at pH 7.0, and (ΔH = −7 kcal/mol, ΔS = −19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory–experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop–stem tertiary contacts from the nucleotide sequence alone. PMID:20100813

Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jia; Yang, Yuting; Wan, Ke

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, ourmore » structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.« less

Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase

PubMed Central

Ohki, Taku; Shibata, Naoki; Higuchi, Yoshiki; Kawashima, Yasuyuki; Takeo, Masahiro; Kato, Dai-ichiro; Negoro, Seiji

2009-01-01

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s−1) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-Oɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold. PMID:19521995

Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: The case of the fold-type I, pyridoxal-5′-phosphate-dependent enzymes

PubMed Central

Paiardini, Alessandro; Bossa, Francesco; Pascarella, Stefano

2004-01-01

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly (r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction. PMID:15498941

Reactions of dipolar bio-molecules in nano-capsules--example of folding-unfolding process.

PubMed

Sanfeld, A; Sefiane, K; Steinchen, A

2011-11-14

The confinement of chemical reactions in nano-capsules can lead to a dramatic effect on the equilibrium constant of these latter. Indeed, capillary effects due to the curvature and surface energy of nano-capsules can alter in a noticeable way the evolution of reactions occurring within. Nano-encapsulation of bio-materials has attracted lately wide interest from the scientific community because of the great potential of its applications in biomedical areas and targeted therapies. The present paper focuses one's attention on alterations of conformation mechanisms due to extremely confining and interacting solvated dipolar macromolecules at their isoelectric point. As a specific example studied here, the folding-unfolding reaction of proteins (particularly RNase A and creatine kinase CK) is drastically changed when encapsulated in solid inorganic hollow nano-capsules. The effects demonstrated in this work can be extended to a wide variety of nano-encapsulation situations. The design and sizing of nano-capsules can even make use of the effects shown in the present study to achieve better and more effective encapsulation. Copyright © 2011 Elsevier B.V. All rights reserved.

The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.

2008-01-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstromsmore » resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.« less

Systematic Validation of Protein Force Fields against Experimental Data

PubMed Central

Eastwood, Michael P.; Dror, Ron O.; Shaw, David E.

2012-01-01

Molecular dynamics simulations provide a vehicle for capturing the structures, motions, and interactions of biological macromolecules in full atomic detail. The accuracy of such simulations, however, is critically dependent on the force field—the mathematical model used to approximate the atomic-level forces acting on the simulated molecular system. Here we present a systematic and extensive evaluation of eight different protein force fields based on comparisons of experimental data with molecular dynamics simulations that reach a previously inaccessible timescale. First, through extensive comparisons with experimental NMR data, we examined the force fields' abilities to describe the structure and fluctuations of folded proteins. Second, we quantified potential biases towards different secondary structure types by comparing experimental and simulation data for small peptides that preferentially populate either helical or sheet-like structures. Third, we tested the force fields' abilities to fold two small proteins—one α-helical, the other with β-sheet structure. The results suggest that force fields have improved over time, and that the most recent versions, while not perfect, provide an accurate description of many structural and dynamical properties of proteins. PMID:22384157

The flower of Hibiscus trionum is both visibly and measurably iridescent.

PubMed

Vignolini, Silvia; Moyroud, Edwige; Hingant, Thomas; Banks, Hannah; Rudall, Paula J; Steiner, Ullrich; Glover, Beverley J

2015-01-01

Living organisms can use minute structures to manipulate the reflection of light and display colours based on interference. There has been debate in recent literature over whether the diffractive optical effects produced by epoxy replicas of petals with folded cuticles persist and induce iridescence in the original flowers when the effects of petal pigment and illumination are taken into account. We explored the optical properties of the petal of Hibiscus trionum by macro-imaging, scanning and transmission electron microscopy, and visible and ultraviolet (UV) angle-resolved spectroscopy of the petal. The flower of Hibiscus trionum is visibly iridescent, and the iridescence can be captured photographically. The iridescence derives from a diffraction grating generated by folds of the cuticle. The iridescence of the petal can be quantitatively characterized by spectrometric measurements with several square-millimetres of sample area illuminated. The flower of Hibiscus trionum has the potential to interact with its pollinators (honeybees, other bees, butterflies and flies) through iridescent signals produced by its cuticular diffraction grating. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Complete fold annotation of the human proteome using a novel structural feature space

DOE PAGES

Middleton, Sarah A.; Illuminati, Joseph; Kim, Junhyong

2017-04-13

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 94% accuracy even when many folds have only one training example. We demonstrate the utility of this methodmore » by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into potential biological function, such as prediction of RNA-binding ability. Finally, our method can be applied to de novo fold prediction of entire proteomes and identify candidate novel fold families.« less

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations

PubMed Central

Matthews, Holly; Deakin, Jon; Rajab, May; Idris-Usman, Maryam

2017-01-01

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26–32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product. PMID:28257497

Two unprecedented aromatic guanidines supramolecular chains self-assembled by hydrogen bonding interaction

NASA Astrophysics Data System (ADS)

Zhang, Yunshen; Huang, Yichao; Zhang, Jiangwei; Zhu, Li; Chen, Kun; Hao, Jian

2015-10-01

Two aromatic guanidine derivatives, C6H5N = C(NHCy)2 (1), (n-TBA)C6H5NHC(NHCy)2Mo2O7 (2) (Cy = cyclohexyl), were synthetized with high yields. Both of them self-assembled into supramolecules via H-bond interactions. Single crystal XRD indicated that crystal 1 showed helix chains combining pseudo four-fold and pseudo six-fold symmetries, while crystal 2 presented ladder chains with alternate ring structures. In this paper, a novel way to design ladder-like supramolecular chains from helix chains was presented, using POMs (polyoxometalates) to provide protons to help assembly.

Determining the folding and binding free energy of DNA-based nanodevices and nanoswitches using urea titration curves

PubMed Central

Idili, Andrea

2017-01-01

Abstract DNA nanotechnology takes advantage of the predictability of DNA interactions to build complex DNA-based functional nanoscale structures. However, when DNA functional and responsive units that are based on non-canonical DNA interactions are employed it becomes quite challenging to predict, understand and control their thermodynamics. In response to this limitation, here we demonstrate the use of isothermal urea titration experiments to estimate the free energy involved in a set of DNA-based systems ranging from unimolecular DNA-based nanoswitches to more complex DNA folds (e.g. aptamers) and nanodevices. We propose here a set of fitting equations that allow to analyze the urea titration curves of these DNA responsive units based on Watson–Crick and non-canonical interactions (stem-loop, G-quadruplex, triplex structures) and to correctly estimate their relative folding and binding free energy values under different experimental conditions. The results described herein will pave the way toward the use of urea titration experiments in the field of DNA nanotechnology to achieve easier and more reliable thermodynamic characterization of DNA-based functional responsive units. More generally, our results will be of general utility to characterize other complex supramolecular systems based on different biopolymers. PMID:28605461

Effects of mucosal loading on vocal fold vibration.

PubMed

Tao, Chao; Jiang, Jack J

2009-06-01

A chain model was proposed in this study to examine the effects of mucosal loading on vocal fold vibration. Mucosal loading was defined as the loading caused by the interaction between the vocal folds and the surrounding tissue. In the proposed model, the vocal folds and the surrounding tissue were represented by a series of oscillators connected by a coupling spring. The lumped masses, springs, and dampers of the oscillators modeled the tissue properties of mass, stiffness, and viscosity, respectively. The coupling spring exemplified the tissue interactions. By numerically solving this chain model, the effects of mucosal loading on the phonation threshold pressure, phonation instability pressure, and energy distribution in a voice production system were studied. It was found that when mucosal loading is small, phonation threshold pressure increases with the damping constant R(r), the mass constant R(m), and the coupling constant R(mu) of mucosal loading but decreases with the stiffness constant R(k). Phonation instability pressure is also related to mucosal loading. It was found that phonation instability pressure increases with the coupling constant R(mu) but decreases with the stiffness constant R(k) of mucosal loading. Therefore, it was concluded that mucosal loading directly affects voice production.

Effects of mucosal loading on vocal fold vibration

NASA Astrophysics Data System (ADS)

Tao, Chao; Jiang, Jack J.

2009-06-01

A chain model was proposed in this study to examine the effects of mucosal loading on vocal fold vibration. Mucosal loading was defined as the loading caused by the interaction between the vocal folds and the surrounding tissue. In the proposed model, the vocal folds and the surrounding tissue were represented by a series of oscillators connected by a coupling spring. The lumped masses, springs, and dampers of the oscillators modeled the tissue properties of mass, stiffness, and viscosity, respectively. The coupling spring exemplified the tissue interactions. By numerically solving this chain model, the effects of mucosal loading on the phonation threshold pressure, phonation instability pressure, and energy distribution in a voice production system were studied. It was found that when mucosal loading is small, phonation threshold pressure increases with the damping constant Rr, the mass constant Rm, and the coupling constant Rμ of mucosal loading but decreases with the stiffness constant Rk. Phonation instability pressure is also related to mucosal loading. It was found that phonation instability pressure increases with the coupling constant Rμ but decreases with the stiffness constant Rk of mucosal loading. Therefore, it was concluded that mucosal loading directly affects voice production.

Principles for computational design of binding antibodies

PubMed Central

Pszolla, M. Gabriele; Lapidoth, Gideon D.; Norn, Christoffer; Dym, Orly; Unger, Tamar; Albeck, Shira; Tyka, Michael D.; Fleishman, Sarel J.

2017-01-01

Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called “ideal” folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes. PMID:28973872

A new topology of the HK97-like fold revealed in Bordetella bacteriophage by cryoEM at 3.5 Å resolution

PubMed Central

Zhang, Xing; Guo, Huatao; Jin, Lei; Czornyj, Elizabeth; Hodes, Asher; Hui, Wong H; Nieh, Angela W; Miller, Jeff F; Zhou, Z Hong

2013-01-01

Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement (DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3.5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein (CP) and HK97-like (‘Johnson’) for its major capsid protein (MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. β-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering. DOI: http://dx.doi.org/10.7554/eLife.01299.001 PMID:24347545

Biochemical, structural and functional diversity between two digestive α-amylases from Helicoverpa armigera.

PubMed

Bhide, Amey J; Channale, Sonal M; Patil, Sucheta S; Gupta, Vidya S; Ramasamy, Sureshkumar; Giri, Ashok P

2015-09-01

Helicoverpa armigera (Lepidoptera) feeds on various plants using diverse digestive enzymes as one of the survival tool-kit. The aim of the present study was to understand biochemical properties of recombinant α-amylases of H. armigera viz., HaAmy1 and HaAmy2. The open reading frames of HaAmy1 and HaAmy2 were cloned in Pichia pastoris and expressed heterologously. Purified recombinant enzymes were characterized for their biochemical and biophysical attributes using established methods. Sequence alignment and homology modeling showed that HaAmy1 and HaAmy2 were conserved in their amino acid sequences and structures. HaAmy1 and HaAmy2 showed optimum activity at 60°C; however, they differed in their optimum pH. Furthermore, HaAmy2 showed higher affinity for starch and amylopectin whereas HaAmy1 had higher catalytic efficiency. HaAmy1 and HaAmy2 were inhibited to the same magnitude by a synthetic amylase inhibitor (acarbose) while wheat amylase inhibitor showed about 2-fold higher inhibition of HaAmy1 than HaAmy2 at pH7 while 6-fold difference at pH11. Interactions of HaAmy1 and HaAmy2 with wheat amylase inhibitor revealed 2:1 stoichiometric ratio and much more complex interaction with HaAmy1. The diversity of amylases in perspective of their biochemical and biophysical properties, and their differential interactions with amylase inhibitors signify the potential role of these enzymes in adaptation of H. armigera on diverse plant diets. Characterization of digestive enzymes of H. armigera provides the molecular basis for the polyphagous nature and thus could assist in designing future strategies for the insect control. Copyright © 2015 Elsevier B.V. All rights reserved.

Dopamine Transporter Genetic Variants and Pesticides in Parkinson’s Disease

PubMed Central

Ritz, Beate R.; Manthripragada, Angelika D.; Costello, Sadie; Lincoln, Sarah J.; Farrer, Matthew J.; Cockburn, Myles; Bronstein, Jeff

2009-01-01

Background Research suggests that independent and joint effects of genetic variability in the dopamine transporter (DAT) locus and pesticides may influence Parkinson’s disease (PD) risk. Materials Methods: In 324 incident PD patients and 334 population controls from our rural California case–control study, we genotyped rs2652510, rs2550956 (for the DAT 5′ clades), and the 3′ variable number of tandem repeats (VNTR). Using geographic information system methods, we determined residential exposure to agricultural maneb and paraquat applications. We also collected occupational pesticide use data. Employing logistic regression, we calculated odds ratios (ORs) for clade diplotypes, VNTR genotype, and number of susceptibility (A clade and 9-repeat) alleles and assessed susceptibility allele–pesticide interactions. Results PD risk was increased separately in DAT A clade diplotype carriers [AA vs. BB: OR = 1.66; 95% confidence interval (CI), 1.08–2.57] and 3′ VNTR 9/9 carriers (9/9 vs. 10/10: OR = 1.8; 95% CI, 0.96–3.57), and our data suggest a gene dosing effect. Importantly, high exposure to paraquat and maneb in carriers of one susceptibility allele increased PD risk 3-fold (OR = 2.99; 95% CI, 0.88–10.2), and in carriers of two or more alleles more than 4-fold (OR = 4.53; 95% CI, 1.70–12.1). We obtained similar results for occupational pesticide measures. Discussion Using two independent pesticide measures, we a) replicated previously reported gene–environment interactions between DAT genetic variants and occupational pesticide exposure in men and b) overcame previous limitations of nonspecific pesticide measures and potential recall bias by employing state records and computer models to estimate residential pesticide exposure. Conclusion Our results suggest that DAT genetic variability and pesticide exposure interact to increase PD risk. PMID:19590691

Proof of principle in a de novo designed protein maquette: an allosterically regulated, charge-activated conformational switch in a tetra-alpha-helix bundle.

PubMed

Grosset, A M; Gibney, B R; Rabanal, F; Moser, C C; Dutton, P L

2001-05-08

New understanding of the engineering and allosteric regulation of natural protein conformational switches (such as those that couple chemical and ionic signals, mechanical force, and electro/chemical free energy for biochemical activation, catalysis, and motion) can be derived from simple de novo designed synthetic protein models (maquettes). We demonstrate proof of principle of both reversible switch action and allosteric regulation in a tetra-alpha-helical bundle protein composed of two identical di-helical subunits containing heme coordinated at a specific position close to the disulfide loop region. Individual bundles assume one of two switch states related by large-scale mechanical changes: a syn-topology (helices of the different subunits parallel) or anti-topology (helices antiparallel). Both the spectral properties of a coproporphyrin probe appended to the loop region and the distance-dependent redox interaction between the hemes identify the topologies. Beginning from a syn-topology, introduction of ferric heme in each subunit (either binding or redox change) shifts the topological balance by 25-50-fold (1.9-2.3 kcal/mol) to an anti-dominance. Charge repulsion between the two internal cationic ferric hemes drives the syn- to anti-switch, as demonstrated in two ways. When fixed in the syn-topology, the second ferric heme binding is 25-80-fold (1.9-2.6 kcal/mol) weaker than the first, and adjacent heme redox potentials are split by 80 mV (1.85 kcal/mol), values that energetically match the shift in topological balance. Allosteric and cooperative regulation of the switch by ionic strength exploits the shielded charge interactions between the two hemes and the exposed, cooperative interactions between the coproporphyrin carboxylates.

Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin.

PubMed

Okochi, Mina; Nomura, Tomoko; Zako, Tamotsu; Arakawa, Takatoshi; Iizuka, Ryo; Ueda, Hiroshi; Funatsu, Takashi; Leroux, Michel; Yohda, Masafumi

2004-07-23

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

PubMed

Hilbert, Manuel; Noga, Akira; Frey, Daniel; Hamel, Virginie; Guichard, Paul; Kraatz, Sebastian H W; Pfreundschuh, Moritz; Hosner, Sarah; Flückiger, Isabelle; Jaussi, Rolf; Wieser, Mara M; Thieltges, Katherine M; Deupi, Xavier; Müller, Daniel J; Kammerer, Richard A; Gönczy, Pierre; Hirono, Masafumi; Steinmetz, Michel O

2016-04-01

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

PubMed

Eichmann, Cédric; Preissler, Steffen; Riek, Roland; Deuerling, Elke

2010-05-18

The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.

Three-dimensional optical reconstruction of vocal fold kinematics using high-speed video with a laser projection system

PubMed Central

Luegmair, Georg; Mehta, Daryush D.; Kobler, James B.; Döllinger, Michael

2015-01-01

Vocal fold kinematics and its interaction with aerodynamic characteristics play a primary role in acoustic sound production of the human voice. Investigating the temporal details of these kinematics using high-speed videoendoscopic imaging techniques has proven challenging in part due to the limitations of quantifying complex vocal fold vibratory behavior using only two spatial dimensions. Thus, we propose an optical method of reconstructing the superior vocal fold surface in three spatial dimensions using a high-speed video camera and laser projection system. Using stereo-triangulation principles, we extend the camera-laser projector method and present an efficient image processing workflow to generate the three-dimensional vocal fold surfaces during phonation captured at 4000 frames per second. Initial results are provided for airflow-driven vibration of an ex vivo vocal fold model in which at least 75% of visible laser points contributed to the reconstructed surface. The method captures the vertical motion of the vocal folds at a high accuracy to allow for the computation of three-dimensional mucosal wave features such as vibratory amplitude, velocity, and asymmetry. PMID:26087485

Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

PubMed Central

Vander Meulen, Kirk A.; Davis, Jared H.; Foster, Trenton R.; Record, M. Thomas; Butcher, Samuel E.

2008-01-01

Summary Little is known about the thermodynamic forces that drive the folding pathways of higher order RNA structure. In this study, we employ calorimetric (ITC and DSC) and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop – receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy-driven and highly stable, with a binding constant (Kobs) of 5.5 × 106 M−1 and enthalpy (ΔHobs°) of −33.8 kcal/mol at 45°C in 20 mM KCl and 2 mM MgCl2. Thus we derive the ΔHobs° for a single tetraloop-receptor interaction to be −16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (ΔCp,obs°) of −0.24 kcal·mol−1·K−1 was determined by ITC. DSC and UV monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally 5–state unfolding pathway, and suggest the observed ΔCp,obs° for the interaction results from a temperature-dependent unbound receptor RNA structure. PMID:18845162

A biorobotic model of the human larynx.

PubMed

Manti, M; Cianchetti, M; Nacci, A; Ursino, F; Laschi, C

2015-08-01

This work focuses on a physical model of the human larynx that replicates its main components and functions. The prototype reproduces the multilayer vocal folds and the ab/adduction movements. In particular, the vocal folds prototype is made with soft materials whose mechanical properties have been obtained to be similar to the natural tissue in terms of viscoelasticity. A computational model was used to study fluid-structure interaction between vocal folds and the airflow. This tool allowed us to make a comparison between theoretical and experimental results. Measurements were performed with this prototype in an experimental platform comprising a controlled air flow, pressure sensors and a high-speed camera for measuring vocal fold vibrations. Data included oscillation frequency at the onset pressure and glottal width. Results show that the combination between vocal fold geometry, mechanical properties and dimensions exhibits an oscillation frequency close to that of the human vocal fold. Moreover, computational results show a high correlation with the experimental one.

Measurement of flow separation in a human vocal folds model

NASA Astrophysics Data System (ADS)

Šidlof, Petr; Doaré, Olivier; Cadot, Olivier; Chaigne, Antoine

2011-07-01

The paper provides experimental data on flow separation from a model of the human vocal folds. Data were measured on a four times scaled physical model, where one vocal fold was fixed and the other oscillated due to fluid-structure interaction. The vocal folds were fabricated from silicone rubber and placed on elastic support in the wall of a transparent wind tunnel. A PIV system was used to visualize the flow fields immediately downstream of the glottis and to measure the velocity fields. From the visualizations, the position of the flow separation point was evaluated using a semiautomatic procedure and plotted for different airflow velocities. The separation point position was quantified relative to the orifice width separately for the left and right vocal folds to account for flow asymmetry. The results indicate that the flow separation point remains close to the narrowest cross-section during most of the vocal fold vibration cycle, but moves significantly further downstream shortly prior to and after glottal closure.

On the role of conformational geometry in protein folding

NASA Astrophysics Data System (ADS)

Du, Rose; Pande, Vijay S.; Grosberg, Alexander Yu.; Tanaka, Toyoichi; Shakhnovich, Eugene

1999-12-01

Using a lattice model of protein folding, we find that once certain native contacts have been formed, folding to the native state is inevitable, even if the only energetic bias in the system is nonspecific, homopolymeric attraction to a collapsed state. These conformations can be quite geometrically unrelated to the native state (with as low as only 53% of the native contacts formed). We demonstrate these results by examining the Monte Carlo kinetics of both heteropolymers under Go interactions and homopolymers, with the folding of both types of polymers to the native state of the heteropolymer. Although we only consider a 48-mer lattice model, our findings shed light on the effects of geometrical restrictions, including those of chain connectivity and steric excluded volume, on protein folding. These effects play a complementary role to that of the rugged energy landscape. In addition, the results of this work can aid in the interpretation of experiments and computer simulations of protein folding performed at elevated temperatures.

The Fold Analysis Challenge: A virtual globe-based educational resource

NASA Astrophysics Data System (ADS)

De Paor, Declan G.; Dordevic, Mladen M.; Karabinos, Paul; Tewksbury, Barbara J.; Whitmeyer, Steven J.

2016-04-01

We present an undergraduate structural geology laboratory exercise using the Google Earth virtual globe with COLLADA models, optionally including an interactive stereographic projection and JavaScript controls. The learning resource challenges students to identify bedding traces and estimate bedding orientation at several locations on a fold, to fit the fold axis and axial plane to stereographic projection data, and to fit a doubly-plunging fold model to the large-scale structure. The chosen fold is the Sheep Mountain Anticline, a Laramide uplift in the Big Horn Basin of Wyoming. We take an education research-based approach, guiding students through three levels of difficulty. The exercise aims to counter common student misconceptions and stumbling blocks regarding penetrative structures. It can be used in preparation for an in-person field trip, for post-trip reinforcement, or as a virtual field experience in an online-only course. Our KML scripts can be easily transferred to other fold structures around the globe.

Balancing energy and entropy: A minimalist model for the characterization of protein folding landscapes

PubMed Central

Das, Payel; Matysiak, Silvina; Clementi, Cecilia

2005-01-01

Coarse-grained models have been extremely valuable in promoting our understanding of protein folding. However, the quantitative accuracy of existing simplified models is strongly hindered either from the complete removal of frustration (as in the widely used Gō-like models) or from the compromise with the minimal frustration principle and/or realistic protein geometry (as in the simple on-lattice models). We present a coarse-grained model that “naturally” incorporates sequence details and energetic frustration into an overall minimally frustrated folding landscape. The model is coupled with an optimization procedure to design the parameters of the protein Hamiltonian to fold into a desired native structure. The application to the study of src-Src homology 3 domain shows that this coarse-grained model contains the main physical-chemical ingredients that are responsible for shaping the folding landscape of this protein. The results illustrate the importance of nonnative interactions and energetic heterogeneity for a quantitative characterization of folding mechanisms. PMID:16006532

Effects of tethering a multistate folding protein to a surface

NASA Astrophysics Data System (ADS)

Wei, Shuai; Knotts, Thomas A.

2011-05-01

Protein/surface interactions are important in a variety of fields and devices, yet fundamental understanding of the relevant phenomena remains fragmented due to resolution limitations of experimental techniques. Molecular simulation has provided useful answers, but such studies have focused on proteins that fold through a two-state process. This study uses simulation to show how surfaces can affect proteins which fold through a multistate process by investigating the folding mechanism of lysozyme (PDB ID: 7LZM). The results demonstrate that in the bulk 7LZM folds through a process with four stable states: the folded state, the unfolded state, and two stable intermediates. The folding mechanism remains the same when the protein is tethered to a surface at most residues; however, in one case the folding mechanism changes in such a way as to eliminate one of the intermediates. An analysis of the molecular configurations shows that tethering at this site is advantageous for protein arrays because the active site is both presented to the bulk phase and stabilized. Taken as a whole, the results offer hope that rational design of protein arrays is possible once the behavior of the protein on the surface is ascertained.

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency.

PubMed

Greene, Dina N; Whitney, Spencer M; Matsumura, Ichiro

2007-06-15

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

A fictitious domain method for fluid/solid interaction applied to the plate folding over the 660 Km depth boundary.

NASA Astrophysics Data System (ADS)

Cerpa, Nestor; Hassani, Riad; Gerbault, Muriel

2014-05-01

A large variety of geodynamical problems involve a mechanical system where a competent body is embedded in a more deformable medium, and hence they can be viewed as belonging to the field of solid/fluid interaction.The lithosphere/asthenosphere interaction in subduction zones is among those kind of problems which are generally difficult to tackle numerically since the immersed (solid) body can be geometrically complex and the surrounding (fluid) medium can thus undergo large deformation. Our work presents a new numerical approach for the study of subduction zones. The lithosphere is modeled as a Maxwell viscoelastic body sinking in the viscous asthenosphere. Both domains are discretized by the Finite Element Method (FEM) and we use a staggered coupling method. The interaction is provided by a non-matching interface method called the Fictitious Domain Method (FDM). We have validated this method with some 2-D benchmarks and examples. Through this numerical coupling method we aim at studying the effect of mantle viscosity on the cyclicity of slab folding on the 660 km depth discontinuity approximated as an impenetrable barrier. Depending on the kinematics condition imposed to the overriding and subducting plates, analog and numerical models have previously shown that cyclicity occurs. The viscosity of the asthenosphere (taken as an isoviscous or a double viscosity-layer fluid) impacts on folding cyclicity and consequently on the slab's dip as well as the stress regime of the overriding plate. In particular, applying far-field plate velocities corresponding to those of the South-American and Nazca plates at present, (4.3 cm/yr and 2.9 cm/yr respectively), we obtain periodic slab folding which is consistent with magmatism and sedimentalogical records. These data report cycles in orogenic growth of the order of 30-40 Myrs, a period that we reproduce when the mantle viscosity ranges in between 3 and 5 x 1020 Pa.s. Moreover, we reproduce episodic development of horizontal subduction induced by cyclic folding and, hence, propose a new explanation for episodes of flat subduction under the South-American plate. We show also preliminary results of 3-D subduction.

Folding and unfolding single RNA molecules under tension

PubMed Central

Woodside, Michael T; García-García, Cuauhtémoc; Block, Steven M

2010-01-01

Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: it allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation. PMID:18786653

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

PubMed Central

Bornert, Olivier; Kühl, Tobias; Bremer, Jeroen; van den Akker, Peter C; Pasmooij, Anna MG; Nyström, Alexander

2016-01-01

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)—a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB. PMID:27157667