enzymes dynamics-based alignment: Topics by Science.gov

Sample records for enzymes dynamics-based alignment

Enzyme activity assays within microstructured optical fibers enabled by automated alignment.

PubMed

Warren-Smith, Stephen C; Nie, Guiying; Schartner, Erik P; Salamonsen, Lois A; Monro, Tanya M

2012-12-01

A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women's health.
The Application of the Weighted k-Partite Graph Problem to the Multiple Alignment for Metabolic Pathways.

PubMed

Chen, Wenbin; Hendrix, William; Samatova, Nagiza F

2017-12-01

The problem of aligning multiple metabolic pathways is one of very challenging problems in computational biology. A metabolic pathway consists of three types of entities: reactions, compounds, and enzymes. Based on similarities between enzymes, Tohsato et al. gave an algorithm for aligning multiple metabolic pathways. However, the algorithm given by Tohsato et al. neglects the similarities among reactions, compounds, enzymes, and pathway topology. How to design algorithms for the alignment problem of multiple metabolic pathways based on the similarity of reactions, compounds, and enzymes? It is a difficult computational problem. In this article, we propose an algorithm for the problem of aligning multiple metabolic pathways based on the similarities among reactions, compounds, enzymes, and pathway topology. First, we compute a weight between each pair of like entities in different input pathways based on the entities' similarity score and topological structure using Ay et al.'s methods. We then construct a weighted k-partite graph for the reactions, compounds, and enzymes. We extract a mapping between these entities by solving the maximum-weighted k-partite matching problem by applying a novel heuristic algorithm. By analyzing the alignment results of multiple pathways in different organisms, we show that the alignments found by our algorithm correctly identify common subnetworks among multiple pathways.
Proteins with similar architecture exhibit similar large-scale dynamic behavior.

PubMed Central

Keskin, O; Jernigan, R L; Bahar, I

2000-01-01

We have investigated the similarities and differences in the computed dynamic fluctuations exhibited by six members of a protein fold family with a coarse-grained Gaussian network model. Specifically, we consider the cofactor binding fragment of CysB; the lysine/arginine/ornithine-binding protein (LAO); the enzyme porphobilinogen deaminase (PBGD); the ribose-binding protein (RBP); the N-terminal lobe of ovotransferrin in apo-form (apo-OVOT); and the leucine/isoleucine/valine-binding protein (LIVBP). All have domains that resemble a Rossmann fold, but there are also some significant differences. Results indicate that similar global dynamic behavior is preserved for the members of a fold family, and that differences usually occur in regions only where specific function is localized. The present work is a computational demonstration that the scaffold of a protein fold may be utilized for diverse purposes. LAO requires a bound ligand before it conforms to the large-scale fluctuation behavior of the three other members of the family, CysB, PBGD, and RBP, all of which contain a substrate (cofactor) at the active site cleft. The dynamics of the ligand-free enzymes LIVBP and apo-OVOT, on the other hand, concur with that of unliganded LAO. The present results suggest that it is possible to construct structure alignments based on dynamic fluctuation behavior. PMID:10733987
Comparative study of structural models of Leishmania donovani and human GDP-mannose pyrophosphorylases.

PubMed

Daligaux, Pierre; Bernadat, Guillaume; Tran, Linh; Cavé, Christian; Loiseau, Philippe M; Pomel, Sébastien; Ha-Duong, Tâp

2016-01-01

Leishmania is the parasite responsible for the neglected disease leishmaniasis. Its virulence and survival require biosynthesis of glycoconjugates, whose guanosine diphospho-d-mannose pyrophosphorylase (GDP-MP) is a key player. However, experimentally resolved structures of this enzyme are still lacking. We herein propose structural models of the GDP-MP from human and Leishmania donovani. Based on a multiple sequences alignment, the models were built with MODELLER and then carefully refined with all atom molecular dynamics simulations in explicit solvent. Their quality was evaluated against several standard criteria, including their ability to bind GDP-mannose assessed by redocking calculations. Special attention was given in this study to interactions of the catalytic site residues with the enzyme substrate and competitive inhibitors, opening the perspective of medicinal chemistry developments. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Advantages of a distant cellulase catalytic base.

PubMed

Burgin, Tucker; Ståhlberg, Jerry; Mayes, Heather B

2018-03-30

The inverting glycoside hydrolase Trichoderma reesei ( Hypocrea jecorina ) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing.

PubMed

Gupta, Vikas; Estrada, April D; Blakley, Ivory; Reid, Rob; Patel, Ketan; Meyer, Mason D; Andersen, Stig Uggerhøj; Brown, Allan F; Lila, Mary Ann; Loraine, Ann E

2015-01-01

Blueberries are a rich source of antioxidants and other beneficial compounds that can protect against disease. Identifying genes involved in synthesis of bioactive compounds could enable the breeding of berry varieties with enhanced health benefits. Toward this end, we annotated a previously sequenced draft blueberry genome assembly using RNA-Seq data from five stages of berry fruit development and ripening. Genome-guided assembly of RNA-Seq read alignments combined with output from ab initio gene finders produced around 60,000 gene models, of which more than half were similar to proteins from other species, typically the grape Vitis vinifera. Comparison of gene models to the PlantCyc database of metabolic pathway enzymes identified candidate genes involved in synthesis of bioactive compounds, including bixin, an apocarotenoid with potential disease-fighting properties, and defense-related cyanogenic glycosides, which are toxic. Cyanogenic glycoside (CG) biosynthetic enzymes were highly expressed in green fruit, and a candidate CG detoxification enzyme was up-regulated during fruit ripening. Candidate genes for ethylene, anthocyanin, and 400 other biosynthetic pathways were also identified. Homology-based annotation using Blast2GO and InterPro assigned Gene Ontology terms to around 15,000 genes. RNA-Seq expression profiling showed that blueberry growth, maturation, and ripening involve dynamic gene expression changes, including coordinated up- and down-regulation of metabolic pathway enzymes and transcriptional regulators. Analysis of RNA-seq alignments identified developmentally regulated alternative splicing, promoter use, and 3' end formation. We report genome sequence, gene models, functional annotations, and RNA-Seq expression data that provide an important new resource enabling high throughput studies in blueberry.
Targeted quantification of functional enzyme dynamics in environmental samples for microbially mediated biogeochemical processes: Targeted quantification of functional enzyme dynamics

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

Li, Minjing; Gao, Yuqian; Qian, Wei-Jun

Microbially mediated biogeochemical processes are catalyzed by enzymes that control the transformation of carbon, nitrogen, and other elements in environment. The dynamic linkage between enzymes and biogeochemical species transformation has, however, rarely been investigated because of the lack of analytical approaches to efficiently and reliably quantify enzymes and their dynamics in soils and sediments. Herein, we developed a signature peptide-based technique for sensitively quantifying dissimilatory and assimilatory enzymes using nitrate-reducing enzymes in a hyporheic zone sediment as an example. Moreover, the measured changes in enzyme concentration were found to correlate with the nitrate reduction rate in a way different frommore » that inferred from biogeochemical models based on biomass or functional genes as surrogates for functional enzymes. This phenomenon has important implications for understanding and modeling the dynamics of microbial community functions and biogeochemical processes in environments. Our results also demonstrate the importance of enzyme quantification for the identification and interrogation of those biogeochemical processes with low metabolite concentrations as a result of faster enzyme-catalyzed consumption of metabolites than their production. The dynamic enzyme behaviors provide a basis for the development of enzyme-based models to describe the relationship between the microbial community and biogeochemical processes.« less
Image Alignment for Multiple Camera High Dynamic Range Microscopy.

PubMed

Eastwood, Brian S; Childs, Elisabeth C

2012-01-09

This paper investigates the problem of image alignment for multiple camera high dynamic range (HDR) imaging. HDR imaging combines information from images taken with different exposure settings. Combining information from multiple cameras requires an alignment process that is robust to the intensity differences in the images. HDR applications that use a limited number of component images require an alignment technique that is robust to large exposure differences. We evaluate the suitability for HDR alignment of three exposure-robust techniques. We conclude that image alignment based on matching feature descriptors extracted from radiant power images from calibrated cameras yields the most accurate and robust solution. We demonstrate the use of this alignment technique in a high dynamic range video microscope that enables live specimen imaging with a greater level of detail than can be captured with a single camera.
Image Alignment for Multiple Camera High Dynamic Range Microscopy

PubMed Central

Eastwood, Brian S.; Childs, Elisabeth C.

2012-01-01

This paper investigates the problem of image alignment for multiple camera high dynamic range (HDR) imaging. HDR imaging combines information from images taken with different exposure settings. Combining information from multiple cameras requires an alignment process that is robust to the intensity differences in the images. HDR applications that use a limited number of component images require an alignment technique that is robust to large exposure differences. We evaluate the suitability for HDR alignment of three exposure-robust techniques. We conclude that image alignment based on matching feature descriptors extracted from radiant power images from calibrated cameras yields the most accurate and robust solution. We demonstrate the use of this alignment technique in a high dynamic range video microscope that enables live specimen imaging with a greater level of detail than can be captured with a single camera. PMID:22545028
Iterative refinement of structure-based sequence alignments by Seed Extension

PubMed Central

Kim, Changhoon; Tai, Chin-Hsien; Lee, Byungkook

2009-01-01

Background Accurate sequence alignment is required in many bioinformatics applications but, when sequence similarity is low, it is difficult to obtain accurate alignments based on sequence similarity alone. The accuracy improves when the structures are available, but current structure-based sequence alignment procedures still mis-align substantial numbers of residues. In order to correct such errors, we previously explored the possibility of replacing the residue-based dynamic programming algorithm in structure alignment procedures with the Seed Extension algorithm, which does not use a gap penalty. Here, we describe a new procedure called RSE (Refinement with Seed Extension) that iteratively refines a structure-based sequence alignment. Results RSE uses SE (Seed Extension) in its core, which is an algorithm that we reported recently for obtaining a sequence alignment from two superimposed structures. The RSE procedure was evaluated by comparing the correctly aligned fractions of residues before and after the refinement of the structure-based sequence alignments produced by popular programs. CE, DaliLite, FAST, LOCK2, MATRAS, MATT, TM-align, SHEBA and VAST were included in this analysis and the NCBI's CDD root node set was used as the reference alignments. RSE improved the average accuracy of sequence alignments for all programs tested when no shift error was allowed. The amount of improvement varied depending on the program. The average improvements were small for DaliLite and MATRAS but about 5% for CE and VAST. More substantial improvements have been seen in many individual cases. The additional computation times required for the refinements were negligible compared to the times taken by the structure alignment programs. Conclusion RSE is a computationally inexpensive way of improving the accuracy of a structure-based sequence alignment. It can be used as a standalone procedure following a regular structure-based sequence alignment or to replace the traditional iterative refinement procedures based on residue-level dynamic programming algorithm in many structure alignment programs. PMID:19589133
Enzyme sequence similarity improves the reaction alignment method for cross-species pathway comparison

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

Ovacik, Meric A.; Androulakis, Ioannis P., E-mail: yannis@rci.rutgers.edu; Biomedical Engineering Department, Rutgers University, Piscataway, NJ 08854

2013-09-15

Pathway-based information has become an important source of information for both establishing evolutionary relationships and understanding the mode of action of a chemical or pharmaceutical among species. Cross-species comparison of pathways can address two broad questions: comparison in order to inform evolutionary relationships and to extrapolate species differences used in a number of different applications including drug and toxicity testing. Cross-species comparison of metabolic pathways is complex as there are multiple features of a pathway that can be modeled and compared. Among the various methods that have been proposed, reaction alignment has emerged as the most successful at predicting phylogeneticmore » relationships based on NCBI taxonomy. We propose an improvement of the reaction alignment method by accounting for sequence similarity in addition to reaction alignment method. Using nine species, including human and some model organisms and test species, we evaluate the standard and improved comparison methods by analyzing glycolysis and citrate cycle pathways conservation. In addition, we demonstrate how organism comparison can be conducted by accounting for the cumulative information retrieved from nine pathways in central metabolism as well as a more complete study involving 36 pathways common in all nine species. Our results indicate that reaction alignment with enzyme sequence similarity results in a more accurate representation of pathway specific cross-species similarities and differences based on NCBI taxonomy.« less
Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics.

PubMed

Bauler, Patricia; Huber, Gary; Leyh, Thomas; McCammon, J Andrew

2010-05-06

Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.
GOSAP: Gene Ontology-Based Semantic Alignment of Biological Pathways.

PubMed

Gamalielsson, Jonas; Olsson, Bjorn

2008-01-01

We present a new method for semantic comparison of biological pathways, aiming to discover evolutionary conservation of pathways between species. Our method uses all three sub-ontologies of Gene Ontology (GO) and a measure of semantic similarity to calculate match scores between gene products. These scores are used for finding local pairwise pathway alignments. This approach has the advantage of being applicable to all types of pathways where nodes are gene products, e.g., regulatory pathways, signalling pathways and metabolic enzyme-to-enzyme pathways. We demonstrate the usefulness of the method using regulatory and metabolic pathways from E. coli and S. cerevisiae as examples.
Local alignment vectors reveal cancer cell-induced ECM fiber remodeling dynamics

PubMed Central

Lee, Byoungkoo; Konen, Jessica; Wilkinson, Scott; Marcus, Adam I.; Jiang, Yi

2017-01-01

Invasive cancer cells interact with the surrounding extracellular matrix (ECM), remodeling ECM fiber network structure by condensing, degrading, and aligning these fibers. We developed a novel local alignment vector analysis method to quantitatively measure collagen fiber alignment as a vector field using Circular Statistics. This method was applied to human non-small cell lung carcinoma (NSCLC) cell lines, embedded as spheroids in a collagen gel. Collagen remodeling was monitored using second harmonic generation imaging under normal conditions and when the LKB1-MARK1 pathway was disrupted through RNAi-based approaches. The results showed that inhibiting LKB1 or MARK1 in NSCLC increases the collagen fiber alignment and captures outward alignment vectors from the tumor spheroid, corresponding to high invasiveness of LKB1 mutant cancer cells. With time-lapse imaging of ECM micro-fiber morphology, the local alignment vector can measure the dynamic signature of invasive cancer cell activity and cell-migration-induced ECM and collagen remodeling and realigning dynamics. PMID:28045069
Implied alignment: a synapomorphy-based multiple-sequence alignment method and its use in cladogram search

NASA Technical Reports Server (NTRS)

Wheeler, Ward C.

2003-01-01

A method to align sequence data based on parsimonious synapomorphy schemes generated by direct optimization (DO; earlier termed optimization alignment) is proposed. DO directly diagnoses sequence data on cladograms without an intervening multiple-alignment step, thereby creating topology-specific, dynamic homology statements. Hence, no multiple-alignment is required to generate cladograms. Unlike general and globally optimal multiple-alignment procedures, the method described here, implied alignment (IA), takes these dynamic homologies and traces them back through a single cladogram, linking the unaligned sequence positions in the terminal taxa via DO transformation series. These "lines of correspondence" link ancestor-descendent states and, when displayed as linearly arrayed columns without hypothetical ancestors, are largely indistinguishable from standard multiple alignment. Since this method is based on synapomorphy, the treatment of certain classes of insertion-deletion (indel) events may be different from that of other alignment procedures. As with all alignment methods, results are dependent on parameter assumptions such as indel cost and transversion:transition ratios. Such an IA could be used as a basis for phylogenetic search, but this would be questionable since the homologies derived from the implied alignment depend on its natal cladogram and any variance, between DO and IA + Search, due to heuristic approach. The utility of this procedure in heuristic cladogram searches using DO and the improvement of heuristic cladogram cost calculations are discussed. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.
Evaluation of the safety performance of highway alignments based on fault tree analysis and safety boundaries.

PubMed

Chen, Yikai; Wang, Kai; Xu, Chengcheng; Shi, Qin; He, Jie; Li, Peiqing; Shi, Ting

2018-05-19

To overcome the limitations of previous highway alignment safety evaluation methods, this article presents a highway alignment safety evaluation method based on fault tree analysis (FTA) and the characteristics of vehicle safety boundaries, within the framework of dynamic modeling of the driver-vehicle-road system. Approaches for categorizing the vehicle failure modes while driving on highways and the corresponding safety boundaries were comprehensively investigated based on vehicle system dynamics theory. Then, an overall crash probability model was formulated based on FTA considering the risks of 3 failure modes: losing steering capability, losing track-holding capability, and rear-end collision. The proposed method was implemented on a highway segment between Bengbu and Nanjing in China. A driver-vehicle-road multibody dynamics model was developed based on the 3D alignments of the Bengbu to Nanjing section of Ning-Luo expressway using Carsim, and the dynamics indices, such as sideslip angle and, yaw rate were obtained. Then, the average crash probability of each road section was calculated with a fixed-length method. Finally, the average crash probability was validated against the crash frequency per kilometer to demonstrate the accuracy of the proposed method. The results of the regression analysis and correlation analysis indicated good consistency between the results of the safety evaluation and the crash data and that it outperformed the safety evaluation methods used in previous studies. The proposed method has the potential to be used in practical engineering applications to identify crash-prone locations and alignment deficiencies on highways in the planning and design phases, as well as those in service.
3D-quantitative structure-activity relationship studies on benzothiadiazepine hydroxamates as inhibitors of tumor necrosis factor-alpha converting enzyme.

PubMed

Murumkar, Prashant R; Giridhar, Rajani; Yadav, Mange Ram

2008-04-01

A set of 29 benzothiadiazepine hydroxamates having selective tumor necrosis factor-alpha converting enzyme inhibitory activity were used to compare the quality and predictive power of 3D-quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices models for the atom-based, centroid/atom-based, data-based, and docked conformer-based alignment. Removal of two outliers from the initial training set of molecules improved the predictivity of models. Among the 3D-quantitative structure-activity relationship models developed using the above four alignments, the database alignment provided the optimal predictive comparative molecular field analysis model for the training set with cross-validated r(2) (q(2)) = 0.510, non-cross-validated r(2) = 0.972, standard error of estimates (s) = 0.098, and F = 215.44 and the optimal comparative molecular similarity indices model with cross-validated r(2) (q(2)) = 0.556, non-cross-validated r(2) = 0.946, standard error of estimates (s) = 0.163, and F = 99.785. These models also showed the best test set prediction for six compounds with predictive r(2) values of 0.460 and 0.535, respectively. The contour maps obtained from 3D-quantitative structure-activity relationship studies were appraised for activity trends for the molecules analyzed. The comparative molecular similarity indices models exhibited good external predictivity as compared with that of comparative molecular field analysis models. The data generated from the present study helped us to further design and report some novel and potent tumor necrosis factor-alpha converting enzyme inhibitors.
Regulation-Structured Dynamic Metabolic Model Provides a Potential Mechanism for Delayed Enzyme Response in Denitrification Process

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

Song, Hyun-Seob; Thomas, Dennis G.; Stegen, James C.

In a recent study of denitrification dynamics in hyporheic zone sediments, we observed a significant time lag (up to several days) in enzymatic response to the changes in substrate concentration. To explore an underlying mechanism and understand the interactive dynamics between enzymes and nutrients, we developed a trait-based model that associates a community’s traits with functional enzymes, instead of typically used species guilds (or functional guilds). This enzyme-based formulation allows to collectively describe biogeochemical functions of microbial communities without directly parameterizing the dynamics of species guilds, therefore being scalable to complex communities. As a key component of modeling, we accountedmore » for microbial regulation occurring through transcriptional and translational processes, the dynamics of which was parameterized based on the temporal profiles of enzyme concentrations measured using a new signature peptide-based method. The simulation results using the resulting model showed several days of a time lag in enzymatic responses as observed in experiments. Further, the model showed that the delayed enzymatic reactions could be primarily controlled by transcriptional responses and that the dynamics of transcripts and enzymes are closely correlated. The developed model can serve as a useful tool for predicting biogeochemical processes in natural environments, either independently or through integration with hydrologic flow simulators.« less
Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis

PubMed Central

Anbar, Michael; Gul, Ozgur; Lamed, Raphael; Sezerman, Ugur O.

2012-01-01

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997–1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A. PMID:22389377
Structure/Function Analyses of Human Serum Paraoxonase (HuPON1) Mutants Designed from a DFPase-Like Homology Model

DTIC Science & Technology

2004-08-23

purified HuPON1 Substitution of amino acid residues in the HuPONI enzyme was accomplished by PCR-based site-directed Two methods were utilized to...including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzyme’s...not been assigned to those residues. Based on scquence-structure alignment studies, we have folded the amino acid sequence of HuPON I onto the sixfold

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

PubMed Central

Gopal, J; Yebra, M J; Bhagwat, A S

1994-01-01

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework. Images PMID:7971279
Invariant glycines and prolines flanking in loops the strand beta 2 of various (alpha/beta)8-barrel enzymes: a hidden homology?

PubMed Central

Janecek, S.

1996-01-01

The question of parallel (alpha/beta)8-barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (alpha/beta)8-barrel enzymes. The "classical" approaches used in the search for homologies among (alpha/beta)8-barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the homologies. These are proposed to be "hidden" in (alpha/beta)8-barrel enzymes. The term "hidden homology" means that the alignment of sequence stretches proposed to be homologous need not be structurally fully satisfactory. This is due to the very long evolutionary history of all (alpha/beta)8-barrels. This work identifies so-called hidden homology around the strand beta 2 that is flanked by loops containing invariant glycines and prolines in 17 different (alpha/beta)8-barrel enzymes, i.e., roughly in half of all currently known (alpha/beta)8-barrel proteins. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif, given their mutual evolutionary relatedness. For this purpose, the sequence region around the well-conserved second beta-strand of alpha-amylase flanked by the invariant glycine and proline (56_GFTAIWITP, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The proposal that the second beta-strand of (alpha/beta)8-barrel fold is important from the evolutionary point of view is strongly supported by the increasing trend of the observed beta 2-strand structural similarity for the pairs of (alpha/beta)8-barrel enzymes: alpha-amylase and the alpha-subunit of tryptophan synthase, alpha-amylase and mandelate racemase, and alpha-amylase and cyclodextrin glycosyltransferase. This trend is also in agreement with the existing evolutionary division of the entire family of (alpha/beta)8-barrel proteins. PMID:8762144
Invariant glycines and prolines flanking in loops the strand beta 2 of various (alpha/beta)8-barrel enzymes: a hidden homology?

PubMed

Janecek, S

1996-06-01

The question of parallel (alpha/beta)8-barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (alpha/beta)8-barrel enzymes. The "classical" approaches used in the search for homologies among (alpha/beta)8-barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the homologies. These are proposed to be "hidden" in (alpha/beta)8-barrel enzymes. The term "hidden homology" means that the alignment of sequence stretches proposed to be homologous need not be structurally fully satisfactory. This is due to the very long evolutionary history of all (alpha/beta)8-barrels. This work identifies so-called hidden homology around the strand beta 2 that is flanked by loops containing invariant glycines and prolines in 17 different (alpha/beta)8-barrel enzymes, i.e., roughly in half of all currently known (alpha/beta)8-barrel proteins. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif, given their mutual evolutionary relatedness. For this purpose, the sequence region around the well-conserved second beta-strand of alpha-amylase flanked by the invariant glycine and proline (56_GFTAIWITP, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The proposal that the second beta-strand of (alpha/beta)8-barrel fold is important from the evolutionary point of view is strongly supported by the increasing trend of the observed beta 2-strand structural similarity for the pairs of (alpha/beta)8-barrel enzymes: alpha-amylase and the alpha-subunit of tryptophan synthase, alpha-amylase and mandelate racemase, and alpha-amylase and cyclodextrin glycosyltransferase. This trend is also in agreement with the existing evolutionary division of the entire family of (alpha/beta)8-barrel proteins.
Effect of alignment of easy axes on dynamic magnetization of immobilized magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Yoshida, Takashi; Matsugi, Yuki; Tsujimura, Naotaka; Sasayama, Teruyoshi; Enpuku, Keiji; Viereck, Thilo; Schilling, Meinhard; Ludwig, Frank

2017-04-01

In some biomedical applications of magnetic nanoparticles (MNPs), the particles are physically immobilized. In this study, we explore the effect of the alignment of the magnetic easy axes on the dynamic magnetization of immobilized MNPs under an AC excitation field. We prepared three immobilized MNP samples: (1) a sample in which easy axes are randomly oriented, (2) a parallel-aligned sample in which easy axes are parallel to the AC field, and (3) an orthogonally aligned sample in which easy axes are perpendicular to the AC field. First, we show that the parallel-aligned sample has the largest hysteresis in the magnetization curve and the largest harmonic magnetization spectra, followed by the randomly oriented and orthogonally aligned samples. For example, 1.6-fold increase was observed in the area of the hysteresis loop of the parallel-aligned sample compared to that of the randomly oriented sample. To quantitatively discuss the experimental results, we perform a numerical simulation based on a Fokker-Planck equation, in which probability distributions for the directions of the easy axes are taken into account in simulating the prepared MNP samples. We obtained quantitative agreement between experiment and simulation. These results indicate that the dynamic magnetization of immobilized MNPs is significantly affected by the alignment of the easy axes.
Clinical Phenotype Classifications Based on Static Varus Alignment and Varus Thrust in Japanese Patients With Medial Knee Osteoarthritis

PubMed Central

Iijima, Hirotaka; Fukutani, Naoto; Fukumoto, Takahiko; Uritani, Daisuke; Kaneda, Eishi; Ota, Kazuo; Kuroki, Hiroshi; Matsuda, Shuichi

2015-01-01

Objective To investigate the association between knee pain during gait and 4 clinical phenotypes based on static varus alignment and varus thrust in patients with medial knee osteoarthritis (OA). Methods Patients in an orthopedic clinic (n = 266) diagnosed as having knee OA (Kellgren/Lawrence [K/L] grade ≥1) were divided into 4 phenotype groups according to the presence or absence of static varus alignment and varus thrust (dynamic varus): no varus (n = 173), dynamic varus (n = 17), static varus (n = 50), and static varus + dynamic varus (n = 26). The knee range of motion, spatiotemporal gait parameters, visual analog scale scores for knee pain, and scores on the Japanese Knee Osteoarthritis Measure were used to assess clinical outcomes. Multiple logistic regression analyses identified the relationship between knee pain during gait and the 4 phenotypes, adjusted for possible risk factors, including age, sex, body mass index, K/L grade, and gait velocity. Results Multiple logistic regression analysis showed that varus thrust without varus alignment was associated with knee pain during gait (odds ratio [OR] 3.30, 95% confidence interval [95% CI] 1.08–12.4), and that varus thrust combined with varus alignment was strongly associated with knee pain during gait (OR 17.1, 95% CI 3.19–320.0). Sensitivity analyses applying alternative cutoff values for defining static varus alignment showed comparable results. Conclusion Varus thrust with or without static varus alignment was associated with the occurrence of knee pain during gait. Tailored interventions based on individual malalignment phenotypes may improve clinical outcomes in patients with knee OA. PMID:26017348
Warp-averaging event-related potentials.

PubMed

Wang, K; Begleiter, H; Porjesz, B

2001-10-01

To align the repeated single trials of the event-related potential (ERP) in order to get an improved estimate of the ERP. A new implementation of the dynamic time warping is applied to compute a warp-average of the single trials. The trilinear modeling method is applied to filter the single trials prior to alignment. Alignment is based on normalized signals and their estimated derivatives. These features reduce the misalignment due to aligning the random alpha waves, explaining amplitude differences in latency differences, or the seemingly small amplitudes of some components. Simulations and applications to visually evoked potentials show significant improvement over some commonly used methods. The new implementation of the dynamic time warping can be used to align the major components (P1, N1, P2, N2, P3) of the repeated single trials. The average of the aligned single trials is an improved estimate of the ERP. This could lead to more accurate results in subsequent analysis.
Local alignment of two-base encoded DNA sequence

PubMed Central

Homer, Nils; Merriman, Barry; Nelson, Stanley F

2009-01-01

Background DNA sequence comparison is based on optimal local alignment of two sequences using a similarity score. However, some new DNA sequencing technologies do not directly measure the base sequence, but rather an encoded form, such as the two-base encoding considered here. In order to compare such data to a reference sequence, the data must be decoded into sequence. The decoding is deterministic, but the possibility of measurement errors requires searching among all possible error modes and resulting alignments to achieve an optimal balance of fewer errors versus greater sequence similarity. Results We present an extension of the standard dynamic programming method for local alignment, which simultaneously decodes the data and performs the alignment, maximizing a similarity score based on a weighted combination of errors and edits, and allowing an affine gap penalty. We also present simulations that demonstrate the performance characteristics of our two base encoded alignment method and contrast those with standard DNA sequence alignment under the same conditions. Conclusion The new local alignment algorithm for two-base encoded data has substantial power to properly detect and correct measurement errors while identifying underlying sequence variants, and facilitating genome re-sequencing efforts based on this form of sequence data. PMID:19508732
A dynamic programming approach for the alignment of signal peaks in multiple gas chromatography-mass spectrometry experiments.

PubMed

Robinson, Mark D; De Souza, David P; Keen, Woon Wai; Saunders, Eleanor C; McConville, Malcolm J; Speed, Terence P; Likić, Vladimir A

2007-10-29

Gas chromatography-mass spectrometry (GC-MS) is a robust platform for the profiling of certain classes of small molecules in biological samples. When multiple samples are profiled, including replicates of the same sample and/or different sample states, one needs to account for retention time drifts between experiments. This can be achieved either by the alignment of chromatographic profiles prior to peak detection, or by matching signal peaks after they have been extracted from chromatogram data matrices. Automated retention time correction is particularly important in non-targeted profiling studies. A new approach for matching signal peaks based on dynamic programming is presented. The proposed approach relies on both peak retention times and mass spectra. The alignment of more than two peak lists involves three steps: (1) all possible pairs of peak lists are aligned, and similarity of each pair of peak lists is estimated; (2) the guide tree is built based on the similarity between the peak lists; (3) peak lists are progressively aligned starting with the two most similar peak lists, following the guide tree until all peak lists are exhausted. When two or more experiments are performed on different sample states and each consisting of multiple replicates, peak lists within each set of replicate experiments are aligned first (within-state alignment), and subsequently the resulting alignments are aligned themselves (between-state alignment). When more than two sets of replicate experiments are present, the between-state alignment also employs the guide tree. We demonstrate the usefulness of this approach on GC-MS metabolic profiling experiments acquired on wild-type and mutant Leishmania mexicana parasites. We propose a progressive method to match signal peaks across multiple GC-MS experiments based on dynamic programming. A sensitive peak similarity function is proposed to balance peak retention time and peak mass spectra similarities. This approach can produce the optimal alignment between an arbitrary number of peak lists, and models explicitly within-state and between-state peak alignment. The accuracy of the proposed method was close to the accuracy of manually-curated peak matching, which required tens of man-hours for the analyzed data sets. The proposed approach may offer significant advantages for processing of high-throughput metabolomics data, especially when large numbers of experimental replicates and multiple sample states are analyzed.
Diffusional correlations among multiple active sites in a single enzyme.

PubMed

Echeverria, Carlos; Kapral, Raymond

2014-04-07

Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.
Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

PubMed Central

2012-01-01

Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants. PMID:22883984
Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia.

PubMed

Zuiter, Afnan Saeid; Sawwan, Jammal; Al Abdallat, Ayed

2012-08-10

Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.
Spreadsheet-based program for alignment of overlapping DNA sequences.

PubMed

Anbazhagan, R; Gabrielson, E

1999-06-01

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.
ChromA: signal-based retention time alignment for chromatography-mass spectrometry data.

PubMed

Hoffmann, Nils; Stoye, Jens

2009-08-15

We describe ChromA, a web-based alignment tool for chromatography-mass spectrometry data from the metabolomics and proteomics domains. Users can supply their data in open and standardized file formats for retention time alignment using dynamic time warping with different configurable local distance and similarity functions. Additionally, user-defined anchors can be used to constrain and speedup the alignment. A neighborhood around each anchor can be added to increase the flexibility of the constrained alignment. ChromA offers different visualizations of the alignment for easier qualitative interpretation and comparison of the data. For the multiple alignment of more than two data files, the center-star approximation is applied to select a reference among input files to align to. ChromA is available at http://bibiserv.techfak.uni-bielefeld.de/chroma. Executables and source code under the L-GPL v3 license are provided for download at the same location.
Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

PubMed

Finnigan, William; Thomas, Adam; Cromar, Holly; Gough, Ben; Snajdrova, Radka; Adams, Joseph P; Littlechild, Jennifer A; Harmer, Nicholas J

2017-03-20

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP + , adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.
EFICAz2: enzyme function inference by a combined approach enhanced by machine learning.

PubMed

Arakaki, Adrian K; Huang, Ying; Skolnick, Jeffrey

2009-04-13

We previously developed EFICAz, an enzyme function inference approach that combines predictions from non-completely overlapping component methods. Two of the four components in the original EFICAz are based on the detection of functionally discriminating residues (FDRs). FDRs distinguish between member of an enzyme family that are homofunctional (classified under the EC number of interest) or heterofunctional (annotated with another EC number or lacking enzymatic activity). Each of the two FDR-based components is associated to one of two specific kinds of enzyme families. EFICAz exhibits high precision performance, except when the maximal test to training sequence identity (MTTSI) is lower than 30%. To improve EFICAz's performance in this regime, we: i) increased the number of predictive components and ii) took advantage of consensual information from the different components to make the final EC number assignment. We have developed two new EFICAz components, analogs to the two FDR-based components, where the discrimination between homo and heterofunctional members is based on the evaluation, via Support Vector Machine models, of all the aligned positions between the query sequence and the multiple sequence alignments associated to the enzyme families. Benchmark results indicate that: i) the new SVM-based components outperform their FDR-based counterparts, and ii) both SVM-based and FDR-based components generate unique predictions. We developed classification tree models to optimally combine the results from the six EFICAz components into a final EC number prediction. The new implementation of our approach, EFICAz2, exhibits a highly improved prediction precision at MTTSI < 30% compared to the original EFICAz, with only a slight decrease in prediction recall. A comparative analysis of enzyme function annotation of the human proteome by EFICAz2 and KEGG shows that: i) when both sources make EC number assignments for the same protein sequence, the assignments tend to be consistent and ii) EFICAz2 generates considerably more unique assignments than KEGG. Performance benchmarks and the comparison with KEGG demonstrate that EFICAz2 is a powerful and precise tool for enzyme function annotation, with multiple applications in genome analysis and metabolic pathway reconstruction. The EFICAz2 web service is available at: http://cssb.biology.gatech.edu/skolnick/webservice/EFICAz2/index.html.
Comparison of Metabolic Pathways in Escherichia coli by Using Genetic Algorithms.

PubMed

Ortegon, Patricia; Poot-Hernández, Augusto C; Perez-Rueda, Ernesto; Rodriguez-Vazquez, Katya

2015-01-01

In order to understand how cellular metabolism has taken its modern form, the conservation and variations between metabolic pathways were evaluated by using a genetic algorithm (GA). The GA approach considered information on the complete metabolism of the bacterium Escherichia coli K-12, as deposited in the KEGG database, and the enzymes belonging to a particular pathway were transformed into enzymatic step sequences by using the breadth-first search algorithm. These sequences represent contiguous enzymes linked to each other, based on their catalytic activities as they are encoded in the Enzyme Commission numbers. In a posterior step, these sequences were compared using a GA in an all-against-all (pairwise comparisons) approach. Individual reactions were chosen based on their measure of fitness to act as parents of offspring, which constitute the new generation. The sequences compared were used to construct a similarity matrix (of fitness values) that was then considered to be clustered by using a k-medoids algorithm. A total of 34 clusters of conserved reactions were obtained, and their sequences were finally aligned with a multiple-sequence alignment GA optimized to align all the reaction sequences included in each group or cluster. From these comparisons, maps associated with the metabolism of similar compounds also contained similar enzymatic step sequences, reinforcing the Patchwork Model for the evolution of metabolism in E. coli K-12, an observation that can be expanded to other organisms, for which there is metabolism information. Finally, our mapping of these reactions is discussed, with illustrations from a particular case.
Comparison of Metabolic Pathways in Escherichia coli by Using Genetic Algorithms

PubMed Central

Ortegon, Patricia; Poot-Hernández, Augusto C.; Perez-Rueda, Ernesto; Rodriguez-Vazquez, Katya

2015-01-01

In order to understand how cellular metabolism has taken its modern form, the conservation and variations between metabolic pathways were evaluated by using a genetic algorithm (GA). The GA approach considered information on the complete metabolism of the bacterium Escherichia coli K-12, as deposited in the KEGG database, and the enzymes belonging to a particular pathway were transformed into enzymatic step sequences by using the breadth-first search algorithm. These sequences represent contiguous enzymes linked to each other, based on their catalytic activities as they are encoded in the Enzyme Commission numbers. In a posterior step, these sequences were compared using a GA in an all-against-all (pairwise comparisons) approach. Individual reactions were chosen based on their measure of fitness to act as parents of offspring, which constitute the new generation. The sequences compared were used to construct a similarity matrix (of fitness values) that was then considered to be clustered by using a k-medoids algorithm. A total of 34 clusters of conserved reactions were obtained, and their sequences were finally aligned with a multiple-sequence alignment GA optimized to align all the reaction sequences included in each group or cluster. From these comparisons, maps associated with the metabolism of similar compounds also contained similar enzymatic step sequences, reinforcing the Patchwork Model for the evolution of metabolism in E. coli K-12, an observation that can be expanded to other organisms, for which there is metabolism information. Finally, our mapping of these reactions is discussed, with illustrations from a particular case. PMID:25973143
Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase.

PubMed

Jha, Santosh Kumar; Ji, Minbiao; Gaffney, Kelly J; Boxer, Steven G

2011-10-04

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ(5)-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.
Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

PubMed Central

Jha, Santosh Kumar; Ji, Minbiao; Gaffney, Kelly J.; Boxer, Steven G.

2011-01-01

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ5-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields. PMID:21949360
CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area.

PubMed

Terashi, Genki; Takeda-Shitaka, Mayuko

2015-01-01

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

Dynamic Aftershock Triggering Correlated with Cyclic Loading in the Slip Direction

NASA Astrophysics Data System (ADS)

Hardebeck, J.

2014-12-01

Dynamic stress changes have been shown to contribute to aftershock triggering, but the physical triggering mechanisms are not fully understood. Some proposed mechanisms are based on dynamic stress loading of the target fault in a direction that encourages earthquake slip (e.g. dynamic Coulomb stress triggering), while other mechanisms are based on fault weakening due to shaking. If dynamic stress loading in the fault slip direction plays a role in aftershock triggering, we would expect to see a relationship between the dynamic stress orientations and the aftershock focal mechanisms. Alternatively, if dynamic stress change triggering functions only through a fault weakening mechanism that is independent of the slip direction of the target fault, no such relationship is expected. I study aftershock sequences of 4 M≥6.7 mainshocks in southern California, and find a small but significant relationship between modeled dynamic stress direction and aftershock focal mechanisms. The mainshock dynamic stress changes have two observed impacts: changing the focal mechanisms in a given location to favor those aligned with the dynamic stress change, and changing the spatial distribution of seismicity to favor locations where the dynamic stress change aligns with the background stress. The aftershock focal mechanisms are significantly more aligned with the dynamic stress changes than the preshock mechanisms for only the first 0.5-1 year following most mainshocks, although for at least 10 years following Hector Mine. Dynamic stress effects on focal mechanisms are best observed at long periods (30-60 sec). Dynamic stress effects are only observed when using metrics based on repeated stress cycling in the same direction, for example considering the dominant stress orientation over the full time series, and not for the peak dynamic stress. These results imply that dynamic aftershock triggering operates at least in part through cyclic loading in the direction of fault slip, although non-directional fault weakening may be important as well. This suggests that the orientation of the dynamic stresses, as well as their amplitude, should be considered in the development of physics-based aftershock forecasting models.
Using structure to explore the sequence alignment space of remote homologs.

PubMed

Kuziemko, Andrew; Honig, Barry; Petrey, Donald

2011-10-01

Protein structure modeling by homology requires an accurate sequence alignment between the query protein and its structural template. However, sequence alignment methods based on dynamic programming (DP) are typically unable to generate accurate alignments for remote sequence homologs, thus limiting the applicability of modeling methods. A central problem is that the alignment that is "optimal" in terms of the DP score does not necessarily correspond to the alignment that produces the most accurate structural model. That is, the correct alignment based on structural superposition will generally have a lower score than the optimal alignment obtained from sequence. Variations of the DP algorithm have been developed that generate alternative alignments that are "suboptimal" in terms of the DP score, but these still encounter difficulties in detecting the correct structural alignment. We present here a new alternative sequence alignment method that relies heavily on the structure of the template. By initially aligning the query sequence to individual fragments in secondary structure elements and combining high-scoring fragments that pass basic tests for "modelability", we can generate accurate alignments within a small ensemble. Our results suggest that the set of sequences that can currently be modeled by homology can be greatly extended.
ARYANA: Aligning Reads by Yet Another Approach

PubMed Central

2014-01-01

Motivation Although there are many different algorithms and software tools for aligning sequencing reads, fast gapped sequence search is far from solved. Strong interest in fast alignment is best reflected in the $106 prize for the Innocentive competition on aligning a collection of reads to a given database of reference genomes. In addition, de novo assembly of next-generation sequencing long reads requires fast overlap-layout-concensus algorithms which depend on fast and accurate alignment. Contribution We introduce ARYANA, a fast gapped read aligner, developed on the base of BWA indexing infrastructure with a completely new alignment engine that makes it significantly faster than three other aligners: Bowtie2, BWA and SeqAlto, with comparable generality and accuracy. Instead of the time-consuming backtracking procedures for handling mismatches, ARYANA comes with the seed-and-extend algorithmic framework and a significantly improved efficiency by integrating novel algorithmic techniques including dynamic seed selection, bidirectional seed extension, reset-free hash tables, and gap-filling dynamic programming. As the read length increases ARYANA's superiority in terms of speed and alignment rate becomes more evident. This is in perfect harmony with the read length trend as the sequencing technologies evolve. The algorithmic platform of ARYANA makes it easy to develop mission-specific aligners for other applications using ARYANA engine. Availability ARYANA with complete source code can be obtained from http://github.com/aryana-aligner PMID:25252881
ARYANA: Aligning Reads by Yet Another Approach.

PubMed

Gholami, Milad; Arbabi, Aryan; Sharifi-Zarchi, Ali; Chitsaz, Hamidreza; Sadeghi, Mehdi

2014-01-01

Although there are many different algorithms and software tools for aligning sequencing reads, fast gapped sequence search is far from solved. Strong interest in fast alignment is best reflected in the $10(6) prize for the Innocentive competition on aligning a collection of reads to a given database of reference genomes. In addition, de novo assembly of next-generation sequencing long reads requires fast overlap-layout-concensus algorithms which depend on fast and accurate alignment. We introduce ARYANA, a fast gapped read aligner, developed on the base of BWA indexing infrastructure with a completely new alignment engine that makes it significantly faster than three other aligners: Bowtie2, BWA and SeqAlto, with comparable generality and accuracy. Instead of the time-consuming backtracking procedures for handling mismatches, ARYANA comes with the seed-and-extend algorithmic framework and a significantly improved efficiency by integrating novel algorithmic techniques including dynamic seed selection, bidirectional seed extension, reset-free hash tables, and gap-filling dynamic programming. As the read length increases ARYANA's superiority in terms of speed and alignment rate becomes more evident. This is in perfect harmony with the read length trend as the sequencing technologies evolve. The algorithmic platform of ARYANA makes it easy to develop mission-specific aligners for other applications using ARYANA engine. ARYANA with complete source code can be obtained from http://github.com/aryana-aligner.
ChromA: signal-based retention time alignment for chromatography–mass spectrometry data

PubMed Central

Hoffmann, Nils; Stoye, Jens

2009-01-01

Summary: We describe ChromA, a web-based alignment tool for chromatography–mass spectrometry data from the metabolomics and proteomics domains. Users can supply their data in open and standardized file formats for retention time alignment using dynamic time warping with different configurable local distance and similarity functions. Additionally, user-defined anchors can be used to constrain and speedup the alignment. A neighborhood around each anchor can be added to increase the flexibility of the constrained alignment. ChromA offers different visualizations of the alignment for easier qualitative interpretation and comparison of the data. For the multiple alignment of more than two data files, the center-star approximation is applied to select a reference among input files to align to. Availability: ChromA is available at http://bibiserv.techfak.uni-bielefeld.de/chroma. Executables and source code under the L-GPL v3 license are provided for download at the same location. Contact: stoye@techfak.uni-bielefeld.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19505941
ASPIC: a novel method to predict the exon-intron structure of a gene that is optimally compatible to a set of transcript sequences.

PubMed

Bonizzoni, Paola; Rizzi, Raffaella; Pesole, Graziano

2005-10-05

Currently available methods to predict splice sites are mainly based on the independent and progressive alignment of transcript data (mostly ESTs) to the genomic sequence. Apart from often being computationally expensive, this approach is vulnerable to several problems--hence the need to develop novel strategies. We propose a method, based on a novel multiple genome-EST alignment algorithm, for the detection of splice sites. To avoid limitations of splice sites prediction (mainly, over-predictions) due to independent single EST alignments to the genomic sequence our approach performs a multiple alignment of transcript data to the genomic sequence based on the combined analysis of all available data. We recast the problem of predicting constitutive and alternative splicing as an optimization problem, where the optimal multiple transcript alignment minimizes the number of exons and hence of splice site observations. We have implemented a splice site predictor based on this algorithm in the software tool ASPIC (Alternative Splicing PredICtion). It is distinguished from other methods based on BLAST-like tools by the incorporation of entirely new ad hoc procedures for accurate and computationally efficient transcript alignment and adopts dynamic programming for the refinement of intron boundaries. ASPIC also provides the minimal set of non-mergeable transcript isoforms compatible with the detected splicing events. The ASPIC web resource is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. Extensive bench marking shows that ASPIC outperforms other existing methods in the detection of novel splicing isoforms and in the minimization of over-predictions. ASPIC also requires a lower computation time for processing a single gene and an EST cluster. The ASPIC web resource is available at http://aspic.algo.disco.unimib.it/aspic-devel/.
Dynamic-template-directed multiscale assembly for large-area coating of highly-aligned conjugated polymer thin films

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

Mohammadi, Erfan; Zhao, Chuankai; Meng, Yifei

Solution processable semiconducting polymers have been under intense investigations due to their diverse applications from printed electronics to biomedical devices. However, controlling the macromolecular assembly across length scales during solution coating remains a key challenge, largely due to the disparity in timescales of polymer assembly and high-throughput printing/coating. Herein we propose the concept of dynamic templating to expedite polymer nucleation and the ensuing assembly process, inspired by biomineralization templates capable of surface reconfiguration. Molecular dynamic simulations reveal that surface reconfigurability is key to promoting template–polymer interactions, thereby lowering polymer nucleation barrier. Employing ionic-liquid-based dynamic template during meniscus-guided coating results inmore » highly aligned, highly crystalline donor-acceptor polymer thin films over large area (41cm 2) and promoted charge transport along both the polymer backbone and the π-π stacking direction in field-effect transistors. We further demonstrate that the charge transport anisotropy can be reversed by tuning the degree of polymer backbone alignment.« less
Dynamic-template-directed multiscale assembly for large-area coating of highly-aligned conjugated polymer thin films

PubMed Central

Mohammadi, Erfan; Zhao, Chuankai; Meng, Yifei; Qu, Ge; Zhang, Fengjiao; Zhao, Xikang; Mei, Jianguo; Zuo, Jian-Min; Shukla, Diwakar; Diao, Ying

2017-01-01

Solution processable semiconducting polymers have been under intense investigations due to their diverse applications from printed electronics to biomedical devices. However, controlling the macromolecular assembly across length scales during solution coating remains a key challenge, largely due to the disparity in timescales of polymer assembly and high-throughput printing/coating. Herein we propose the concept of dynamic templating to expedite polymer nucleation and the ensuing assembly process, inspired by biomineralization templates capable of surface reconfiguration. Molecular dynamic simulations reveal that surface reconfigurability is key to promoting template–polymer interactions, thereby lowering polymer nucleation barrier. Employing ionic-liquid-based dynamic template during meniscus-guided coating results in highly aligned, highly crystalline donor–acceptor polymer thin films over large area (>1 cm2) and promoted charge transport along both the polymer backbone and the π–π stacking direction in field-effect transistors. We further demonstrate that the charge transport anisotropy can be reversed by tuning the degree of polymer backbone alignment. PMID:28703136
Dynamic-template-directed multiscale assembly for large-area coating of highly-aligned conjugated polymer thin films

DOE PAGES

Mohammadi, Erfan; Zhao, Chuankai; Meng, Yifei; ...

2017-07-13

Solution processable semiconducting polymers have been under intense investigations due to their diverse applications from printed electronics to biomedical devices. However, controlling the macromolecular assembly across length scales during solution coating remains a key challenge, largely due to the disparity in timescales of polymer assembly and high-throughput printing/coating. Herein we propose the concept of dynamic templating to expedite polymer nucleation and the ensuing assembly process, inspired by biomineralization templates capable of surface reconfiguration. Molecular dynamic simulations reveal that surface reconfigurability is key to promoting template–polymer interactions, thereby lowering polymer nucleation barrier. Employing ionic-liquid-based dynamic template during meniscus-guided coating results inmore » highly aligned, highly crystalline donor-acceptor polymer thin films over large area (41cm 2) and promoted charge transport along both the polymer backbone and the π-π stacking direction in field-effect transistors. We further demonstrate that the charge transport anisotropy can be reversed by tuning the degree of polymer backbone alignment.« less
GATA: A graphic alignment tool for comparative sequenceanalysis

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

Nix, David A.; Eisen, Michael B.

2005-01-01

Several problems exist with current methods used to align DNA sequences for comparative sequence analysis. Most dynamic programming algorithms assume that conserved sequence elements are collinear. This assumption appears valid when comparing orthologous protein coding sequences. Functional constraints on proteins provide strong selective pressure against sequence inversions, and minimize sequence duplications and feature shuffling. For non-coding sequences this collinearity assumption is often invalid. For example, enhancers contain clusters of transcription factor binding sites that change in number, orientation, and spacing during evolution yet the enhancer retains its activity. Dotplot analysis is often used to estimate non-coding sequence relatedness. Yet dotmore » plots do not actually align sequences and thus cannot account well for base insertions or deletions. Moreover, they lack an adequate statistical framework for comparing sequence relatedness and are limited to pairwise comparisons. Lastly, dot plots and dynamic programming text outputs fail to provide an intuitive means for visualizing DNA alignments.« less
Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases.

PubMed

Janecek, S

1994-09-01

Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases.
Alignment of dynamic networks.

PubMed

Vijayan, V; Critchlow, D; Milenkovic, T

2017-07-15

Network alignment (NA) aims to find a node mapping that conserves similar regions between compared networks. NA is applicable to many fields, including computational biology, where NA can guide the transfer of biological knowledge from well- to poorly-studied species across aligned network regions. Existing NA methods can only align static networks. However, most complex real-world systems evolve over time and should thus be modeled as dynamic networks. We hypothesize that aligning dynamic network representations of evolving systems will produce superior alignments compared to aligning the systems' static network representations, as is currently done. For this purpose, we introduce the first ever dynamic NA method, DynaMAGNA ++. This proof-of-concept dynamic NA method is an extension of a state-of-the-art static NA method, MAGNA++. Even though both MAGNA++ and DynaMAGNA++ optimize edge as well as node conservation across the aligned networks, MAGNA++ conserves static edges and similarity between static node neighborhoods, while DynaMAGNA++ conserves dynamic edges (events) and similarity between evolving node neighborhoods. For this purpose, we introduce the first ever measure of dynamic edge conservation and rely on our recent measure of dynamic node conservation. Importantly, the two dynamic conservation measures can be optimized with any state-of-the-art NA method and not just MAGNA++. We confirm our hypothesis that dynamic NA is superior to static NA, on synthetic and real-world networks, in computational biology and social domains. DynaMAGNA++ is parallelized and has a user-friendly graphical interface. http://nd.edu/∼cone/DynaMAGNA++/ . tmilenko@nd.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
Alignment of dynamic networks

PubMed Central

Vijayan, V.; Critchlow, D.; Milenković, T.

2017-01-01

Abstract Motivation: Network alignment (NA) aims to find a node mapping that conserves similar regions between compared networks. NA is applicable to many fields, including computational biology, where NA can guide the transfer of biological knowledge from well- to poorly-studied species across aligned network regions. Existing NA methods can only align static networks. However, most complex real-world systems evolve over time and should thus be modeled as dynamic networks. We hypothesize that aligning dynamic network representations of evolving systems will produce superior alignments compared to aligning the systems’ static network representations, as is currently done. Results: For this purpose, we introduce the first ever dynamic NA method, DynaMAGNA ++. This proof-of-concept dynamic NA method is an extension of a state-of-the-art static NA method, MAGNA++. Even though both MAGNA++ and DynaMAGNA++ optimize edge as well as node conservation across the aligned networks, MAGNA++ conserves static edges and similarity between static node neighborhoods, while DynaMAGNA++ conserves dynamic edges (events) and similarity between evolving node neighborhoods. For this purpose, we introduce the first ever measure of dynamic edge conservation and rely on our recent measure of dynamic node conservation. Importantly, the two dynamic conservation measures can be optimized with any state-of-the-art NA method and not just MAGNA++. We confirm our hypothesis that dynamic NA is superior to static NA, on synthetic and real-world networks, in computational biology and social domains. DynaMAGNA++ is parallelized and has a user-friendly graphical interface. Availability and implementation: http://nd.edu/∼cone/DynaMAGNA++/. Contact: tmilenko@nd.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28881980
Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

DOE PAGES

Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.; ...

2017-01-27

Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less
Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

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

Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.

Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less
A novel approach to multiple sequence alignment using hadoop data grids.

PubMed

Sudha Sadasivam, G; Baktavatchalam, G

2010-01-01

Multiple alignment of protein sequences helps to determine evolutionary linkage and to predict molecular structures. The factors to be considered while aligning multiple sequences are speed and accuracy of alignment. Although dynamic programming algorithms produce accurate alignments, they are computation intensive. In this paper we propose a time efficient approach to sequence alignment that also produces quality alignment. The dynamic nature of the algorithm coupled with data and computational parallelism of hadoop data grids improves the accuracy and speed of sequence alignment. The principle of block splitting in hadoop coupled with its scalability facilitates alignment of very large sequences.
Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

NASA Astrophysics Data System (ADS)

Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti

2016-08-01

Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.
Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes.

PubMed

Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti

2016-08-28

Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.
Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

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

Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu

Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kineticsmore » resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.« less
Initial Alignment for SINS Based on Pseudo-Earth Frame in Polar Regions.

PubMed

Gao, Yanbin; Liu, Meng; Li, Guangchun; Guang, Xingxing

2017-06-16

An accurate initial alignment must be required for inertial navigation system (INS). The performance of initial alignment directly affects the following navigation accuracy. However, the rapid convergence of meridians and the small horizontalcomponent of rotation of Earth make the traditional alignment methods ineffective in polar regions. In this paper, from the perspective of global inertial navigation, a novel alignment algorithm based on pseudo-Earth frame and backward process is proposed to implement the initial alignment in polar regions. Considering that an accurate coarse alignment of azimuth is difficult to obtain in polar regions, the dynamic error modeling with large azimuth misalignment angle is designed. At the end of alignment phase, the strapdown attitude matrix relative to local geographic frame is obtained without influence of position errors and cumbersome computation. As a result, it would be more convenient to access the following polar navigation system. Then, it is also expected to unify the polar alignment algorithm as much as possible, thereby further unifying the form of external reference information. Finally, semi-physical static simulation and in-motion tests with large azimuth misalignment angle assisted by unscented Kalman filter (UKF) validate the effectiveness of the proposed method.

Piezoelectric Response of Aligned Electrospun Polyvinylidene Fluoride/Carbon Nanotube Nanofibrous Membranes.

PubMed

Wu, Chang-Mou; Chou, Min-Hui; Zeng, Wun-Yuan

2018-06-10

Polyvinylidene fluoride (PVDF) shows piezoelectricity related to its β-phase content and mechanical and electrical properties influenced by its morphology and crystallinity. Electrospinning (ES) can produce ultrafine and well-aligned PVDF nanofibers. In this study, the effects of the presence of carbon nanotubes (CNT) and optimized ES parameters on the crystal structures and piezoelectric properties of aligned PVDF/CNT nanofibrous membranes were examined. The optimal β content and piezoelectric coefficient (d 33 ) of the aligned electrospun PVDF reached 88% and 27.4 pC/N; CNT addition increased the β-phase content to 89% and d 33 to 31.3 pC/N. The output voltages of piezoelectric units with aligned electrospun PVDF/CNT membranes increased linearly with applied loading and showed good stability during cyclic dynamic compression and tension. The sensitivities of the piezoelectric units with the membranes under dynamic compression and tension were 2.26 mV/N and 4.29 mV/%, respectively. In bending tests, the output voltage increased nonlinearly with bending angle because complicated forces were involved. The output of the aligned membrane-based piezoelectric unit with CNT was 1.89 V at the bending angle of 100°. The high electric outputs indicate that the aligned electrospun PVDF/CNT membranes are potentially effective for flexible wearable sensor application with high sensitivity.
Alignment-free genetic sequence comparisons: a review of recent approaches by word analysis.

PubMed

Bonham-Carter, Oliver; Steele, Joe; Bastola, Dhundy

2014-11-01

Modern sequencing and genome assembly technologies have provided a wealth of data, which will soon require an analysis by comparison for discovery. Sequence alignment, a fundamental task in bioinformatics research, may be used but with some caveats. Seminal techniques and methods from dynamic programming are proving ineffective for this work owing to their inherent computational expense when processing large amounts of sequence data. These methods are prone to giving misleading information because of genetic recombination, genetic shuffling and other inherent biological events. New approaches from information theory, frequency analysis and data compression are available and provide powerful alternatives to dynamic programming. These new methods are often preferred, as their algorithms are simpler and are not affected by synteny-related problems. In this review, we provide a detailed discussion of computational tools, which stem from alignment-free methods based on statistical analysis from word frequencies. We provide several clear examples to demonstrate applications and the interpretations over several different areas of alignment-free analysis such as base-base correlations, feature frequency profiles, compositional vectors, an improved string composition and the D2 statistic metric. Additionally, we provide detailed discussion and an example of analysis by Lempel-Ziv techniques from data compression. © The Author 2013. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.
PARTS: Probabilistic Alignment for RNA joinT Secondary structure prediction

PubMed Central

Harmanci, Arif Ozgun; Sharma, Gaurav; Mathews, David H.

2008-01-01

A novel method is presented for joint prediction of alignment and common secondary structures of two RNA sequences. The joint consideration of common secondary structures and alignment is accomplished by structural alignment over a search space defined by the newly introduced motif called matched helical regions. The matched helical region formulation generalizes previously employed constraints for structural alignment and thereby better accommodates the structural variability within RNA families. A probabilistic model based on pseudo free energies obtained from precomputed base pairing and alignment probabilities is utilized for scoring structural alignments. Maximum a posteriori (MAP) common secondary structures, sequence alignment and joint posterior probabilities of base pairing are obtained from the model via a dynamic programming algorithm called PARTS. The advantage of the more general structural alignment of PARTS is seen in secondary structure predictions for the RNase P family. For this family, the PARTS MAP predictions of secondary structures and alignment perform significantly better than prior methods that utilize a more restrictive structural alignment model. For the tRNA and 5S rRNA families, the richer structural alignment model of PARTS does not offer a benefit and the method therefore performs comparably with existing alternatives. For all RNA families studied, the posterior probability estimates obtained from PARTS offer an improvement over posterior probability estimates from a single sequence prediction. When considering the base pairings predicted over a threshold value of confidence, the combination of sensitivity and positive predictive value is superior for PARTS than for the single sequence prediction. PARTS source code is available for download under the GNU public license at http://rna.urmc.rochester.edu. PMID:18304945
Whole-proteome phylogeny of large dsDNA viruses and parvoviruses through a composition vector method related to dynamical language model

PubMed Central

2010-01-01

Background The vast sequence divergence among different virus groups has presented a great challenge to alignment-based analysis of virus phylogeny. Due to the problems caused by the uncertainty in alignment, existing tools for phylogenetic analysis based on multiple alignment could not be directly applied to the whole-genome comparison and phylogenomic studies of viruses. There has been a growing interest in alignment-free methods for phylogenetic analysis using complete genome data. Among the alignment-free methods, a dynamical language (DL) method proposed by our group has successfully been applied to the phylogenetic analysis of bacteria and chloroplast genomes. Results In this paper, the DL method is used to analyze the whole-proteome phylogeny of 124 large dsDNA viruses and 30 parvoviruses, two data sets with large difference in genome size. The trees from our analyses are in good agreement to the latest classification of large dsDNA viruses and parvoviruses by the International Committee on Taxonomy of Viruses (ICTV). Conclusions The present method provides a new way for recovering the phylogeny of large dsDNA viruses and parvoviruses, and also some insights on the affiliation of a number of unclassified viruses. In comparison, some alignment-free methods such as the CV Tree method can be used for recovering the phylogeny of large dsDNA viruses, but they are not suitable for resolving the phylogeny of parvoviruses with a much smaller genome size. PMID:20565983
Alignment-free genetic sequence comparisons: a review of recent approaches by word analysis

PubMed Central

Steele, Joe; Bastola, Dhundy

2014-01-01

Modern sequencing and genome assembly technologies have provided a wealth of data, which will soon require an analysis by comparison for discovery. Sequence alignment, a fundamental task in bioinformatics research, may be used but with some caveats. Seminal techniques and methods from dynamic programming are proving ineffective for this work owing to their inherent computational expense when processing large amounts of sequence data. These methods are prone to giving misleading information because of genetic recombination, genetic shuffling and other inherent biological events. New approaches from information theory, frequency analysis and data compression are available and provide powerful alternatives to dynamic programming. These new methods are often preferred, as their algorithms are simpler and are not affected by synteny-related problems. In this review, we provide a detailed discussion of computational tools, which stem from alignment-free methods based on statistical analysis from word frequencies. We provide several clear examples to demonstrate applications and the interpretations over several different areas of alignment-free analysis such as base–base correlations, feature frequency profiles, compositional vectors, an improved string composition and the D2 statistic metric. Additionally, we provide detailed discussion and an example of analysis by Lempel–Ziv techniques from data compression. PMID:23904502
TIM Barrel Protein Structure Classification Using Alignment Approach and Best Hit Strategy

NASA Astrophysics Data System (ADS)

Chu, Jia-Han; Lin, Chun Yuan; Chang, Cheng-Wen; Lee, Chihan; Yang, Yuh-Shyong; Tang, Chuan Yi

2007-11-01

The classification of protein structures is essential for their function determination in bioinformatics. It has been estimated that around 10% of all known enzymes have TIM barrel domains from the Structural Classification of Proteins (SCOP) database. With its high sequence variation and diverse functionalities, TIM barrel protein becomes to be an attractive target for protein engineering and for the evolution study. Hence, in this paper, an alignment approach with the best hit strategy is proposed to classify the TIM barrel protein structure in terms of superfamily and family levels in the SCOP. This work is also used to do the classification for class level in the Enzyme nomenclature (ENZYME) database. Two testing data sets, TIM40D and TIM95D, both are used to evaluate this approach. The resulting classification has an overall prediction accuracy rate of 90.3% for the superfamily level in the SCOP, 89.5% for the family level in the SCOP and 70.1% for the class level in the ENZYME. These results demonstrate that the alignment approach with the best hit strategy is a simple and viable method for the TIM barrel protein structure classification, even only has the amino acid sequences information.
Identification of the srtC1 Transcription Start Site and Catalytically Essential Residues Required for Actinomyces oris T14V SrtC1 Activity

DTIC Science & Technology

2011-07-27

domain (type 2 phosphatidic acid phosphatase) and may be a PAP2 like superfamily member. In order to localize the promoter(s) for these three genes...Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 which amino acid residue(s) was critical for the enzyme activity. This enzyme possesses a...analyzed the role of eight conserved amino acid residues. The amino acids to be mutated were chosen based on the sequence alignment of several class C
Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

PubMed

Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

2017-10-01

Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding†

PubMed Central

Clift, Michael; Ji, Haitao; Deniau, Gildas P.; O’Hagan, David; Silverman, Richard B.

2008-01-01

γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5’-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5’-phosphate (PLP) to pyridoxamine 5’-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred based on the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches. PMID:17988152
Modeling nitrous oxide production and reduction in soil through explicit representation of denitrification enzyme kinetics.

PubMed

Zheng, Jianqiu; Doskey, Paul V

2015-02-17

An enzyme-explicit denitrification model with representations for pre- and de novo synthesized enzymes was developed to improve predictions of nitrous oxide (N2O) accumulations in soil and emissions from the surface. The metabolic model of denitrification is based on dual-substrate utilization and Monod growth kinetics. Enzyme synthesis/activation was incorporated into each sequential reduction step of denitrification to regulate dynamics of the denitrifier population and the active enzyme pool, which controlled the rate function. Parameterizations were developed from observations of the dynamics of N2O production and reduction in soil incubation experiments. The model successfully reproduced the dynamics of N2O and N2 accumulation in the incubations and revealed an important regulatory effect of denitrification enzyme kinetics on the accumulation of denitrification products. Pre-synthesized denitrification enzymes contributed 20, 13, 43, and 62% of N2O that accumulated in 48 h incubations of soil collected from depths of 0-5, 5-10, 10-15, and 15-25 cm, respectively. An enzyme activity function (E) was defined to estimate the relative concentration of active enzymes and variation in response to environmental conditions. The value of E allows for activities of pre-synthesized denitrification enzymes to be differentiated from de novo synthesized enzymes. Incorporating explicit representations of denitrification enzyme kinetics into biogeochemical models is a promising approach for accurately simulating dynamics of the production and reduction of N2O in soils.
Independent Metrics for Protein Backbone and Side-Chain Flexibility: Time Scales and Effects of Ligand Binding.

PubMed

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

2015-03-10

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

PubMed

Lebiedz, Dirk; Kammerer, Julia; Brandt-Pollmann, Ulrich

2005-10-01

We introduce a numerical complexity reduction method for the automatic identification and analysis of dynamic network decompositions in (bio)chemical kinetics based on error-controlled computation of a minimal model dimension represented by the number of (locally) active dynamical modes. Our algorithm exploits a generalized sensitivity analysis along state trajectories and subsequent singular value decomposition of sensitivity matrices for the identification of these dominant dynamical modes. It allows for a dynamic coupling analysis of (bio)chemical species in kinetic models that can be exploited for the piecewise computation of a minimal model on small time intervals and offers valuable functional insight into highly nonlinear reaction mechanisms and network dynamics. We present results for the identification of network decompositions in a simple oscillatory chemical reaction, time scale separation based model reduction in a Michaelis-Menten enzyme system and network decomposition of a detailed model for the oscillatory peroxidase-oxidase enzyme system.
Disparate ultrafast dynamics of itinerant and localized magnetic moments in gadolinium metal

PubMed Central

Frietsch, B.; Bowlan, J.; Carley, R.; Teichmann, M.; Wienholdt, S.; Hinzke, D.; Nowak, U.; Carva, K.; Oppeneer, P. M.; Weinelt, M.

2015-01-01

The Heisenberg–Dirac intra-atomic exchange coupling is responsible for the formation of the atomic spin moment and thus the strongest interaction in magnetism. Therefore, it is generally assumed that intra-atomic exchange leads to a quasi-instantaneous aligning process in the magnetic moment dynamics of spins in separate, on-site atomic orbitals. Following ultrashort optical excitation of gadolinium metal, we concurrently record in photoemission the 4f magnetic linear dichroism and 5d exchange splitting. Their dynamics differ by one order of magnitude, with decay constants of 14 versus 0.8 ps, respectively. Spin dynamics simulations based on an orbital-resolved Heisenberg Hamiltonian combined with first-principles calculations explain the particular dynamics of 5d and 4f spin moments well, and corroborate that the 5d exchange splitting traces closely the 5d spin-moment dynamics. Thus gadolinium shows disparate dynamics of the localized 4f and the itinerant 5d spin moments, demonstrating a breakdown of their intra-atomic exchange alignment on a picosecond timescale. PMID:26355196
Confinement and Structural Changes in Vertically Aligned Dust Structures

NASA Astrophysics Data System (ADS)

Hyde, Truell

2013-10-01

In physics, confinement is known to influence collective system behavior. Examples include coulomb crystal variants such as those formed from ions or dust particles (classical), electrons in quantum dots (quantum) and the structural changes observed in vertically aligned dust particle systems formed within a glass box placed on the lower electrode of a Gaseous Electronics Conference (GEC) rf reference cell. Recent experimental studies have expanded the above to include the biological domain by showing that the stability and dynamics of proteins confined through encapsulation and enzyme molecules placed in inorganic cavities such as those found in biosensors are also directly influenced by their confinement. In this paper, the self-assembly and subsequent collective behavior of structures formed from n, charged dust particles interacting with one another and located within a glass box placed on the lower, powered electrode of a GEC rf reference cell is discussed. Self-organized formation of vertically aligned one-dimensional chains, two-dimensional zigzag structures, and three-dimensional helical structures of triangular, quadrangular, pentagonal, hexagonal, and heptagonal symmetries are shown to occur. System evolution is shown to progress from one-dimensional chain structures, through a zigzag transition to a two-dimensional, spindle like structures, and then to various three-dimensional, helical structures exhibiting various symmetries. Stable configurations are shown to be strongly dependent upon system confinement. The critical conditions for structural transitions as well as the basic symmetry exhibited by the one-, two-, and three-dimensional structures that subsequently develop will be shown to be in good agreement with molecular dynamics simulations.
E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft.

PubMed

Valimberti, Ilaria; Tiberti, Matteo; Lambrughi, Matteo; Sarcevic, Boris; Papaleo, Elena

2015-10-14

Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.
Carbon Nanotubes (CNTs) for the Development of Electrochemical Biosensors

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

Lin, Yuehe; Yantasee, Wassana; Wang, Joseph

2005-01-01

Carbon nanotube (CNT) is a very attractive material for the development of biosensors because of its capability to provide strong electrocatalytic activity and minimize surface fouling of the sensors. This article reviews our recent developments of oxidase- and dehydrogenase-amperometric biosensors based on the immobilization of CNTs, the co-immobilization of enzymes on the CNTs/Nafion or the CNT/Teflon composite materials, or the attachment of enzymes on the controlled-density aligned CNT-nanoelectrode arrays. The excellent electrocatalytic activities of the CNTs on the redox reactions of hydrogen peroxide, nicotinamide adenine dinucleotide (NADH), and homocysteine have been demonstrated. Successful applications of the CNT-based biosensors reviewed hereinmore » include the low-potential detections of glucose, organophosphorus compounds, and alcohol.« less
Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

NASA Astrophysics Data System (ADS)

Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

2016-04-01

Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.
Functional enzyme-based modeling approach for dynamic simulation of denitrification process in hyporheic zone sediments: Genetically structured microbial community model

NASA Astrophysics Data System (ADS)

Song, H. S.; Li, M.; Qian, W.; Song, X.; Chen, X.; Scheibe, T. D.; Fredrickson, J.; Zachara, J. M.; Liu, C.

2016-12-01

Modeling environmental microbial communities at individual organism level is currently intractable due to overwhelming structural complexity. Functional guild-based approaches alleviate this problem by lumping microorganisms into fewer groups based on their functional similarities. This reduction may become ineffective, however, when individual species perform multiple functions as environmental conditions vary. In contrast, the functional enzyme-based modeling approach we present here describes microbial community dynamics based on identified functional enzymes (rather than individual species or their groups). Previous studies in the literature along this line used biomass or functional genes as surrogate measures of enzymes due to the lack of analytical methods for quantifying enzymes in environmental samples. Leveraging our recent development of a signature peptide-based technique enabling sensitive quantification of functional enzymes in environmental samples, we developed a genetically structured microbial community model (GSMCM) to incorporate enzyme concentrations and various other omics measurements (if available) as key modeling input. We formulated the GSMCM based on the cybernetic metabolic modeling framework to rationally account for cellular regulation without relying on empirical inhibition kinetics. In the case study of modeling denitrification process in Columbia River hyporheic zone sediments collected from the Hanford Reach, our GSMCM provided a quantitative fit to complex experimental data in denitrification, including the delayed response of enzyme activation to the change in substrate concentration. Our future goal is to extend the modeling scope to the prediction of carbon and nitrogen cycles and contaminant fate. Integration of a simpler version of the GSMCM with PFLOTRAN for multi-scale field simulations is in progress.
Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in glycoside hydrolase family 1.

PubMed Central

Marques, Ana Rita; Coutinho, Pedro M; Videira, Paula; Fialho, Arsénio M; Sá-Correia, Isabel

2003-01-01

The Sphingomonas paucimobilis beta-glucosidase Bgl1 is encoded by the bgl1 gene, associated with an 1308 bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bgl1 protein through the inner membrane to the periplasmic space. A His(6)-tagged 44.3 kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bgl1 gene lacking the region encoding the putative signal peptide. Mature beta-glucosidase Bgl1 is specific for aryl-beta-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bgl1 and other members of the glycoside hydrolase (GH) family 1 enzymes. At subsite -1, the conserved residues required for catalysis by GH1 enzymes are present in Bgl1 with only minor differences. Major differences are found at subsite +1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH1 enzymes, revealing an absence of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bgl1, the first characterized beta-glucosidase from the genus Sphingomonas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin. PMID:12444924
Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

PubMed Central

Gabba, Matteo; Poblete, Simón; Rosenkranz, Tobias; Katranidis, Alexandros; Kempe, Daryan; Züchner, Tina; Winkler, Roland G.; Gompper, Gerhard; Fitter, Jörg

2014-01-01

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions. PMID:25418172

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

PubMed Central

Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying

2016-01-01

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. PMID:26801571
Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus.

PubMed

Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying; Li, De-Feng; Liu, Zhi-Pei

2016-01-22

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Polyester synthases: natural catalysts for plastics.

PubMed Central

Rehm, Bernd H A

2003-01-01

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism. PMID:12954080
LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations.

PubMed

Schmidt, Thomas H; Kandt, Christian

2012-10-22

At the beginning of each molecular dynamics membrane simulation stands the generation of a suitable starting structure which includes the working steps of aligning membrane and protein and seamlessly accommodating the protein in the membrane. Here we introduce two efficient and complementary methods based on pre-equilibrated membrane patches, automating these steps. Using a voxel-based cast of the coarse-grained protein, LAMBADA computes a hydrophilicity profile-derived scoring function based on which the optimal rotation and translation operations are determined to align protein and membrane. Employing an entirely geometrical approach, LAMBADA is independent from any precalculated data and aligns even large membrane proteins within minutes on a regular workstation. LAMBADA is the first tool performing the entire alignment process automatically while providing the user with the explicit 3D coordinates of the aligned protein and membrane. The second tool is an extension of the InflateGRO method addressing the shortcomings of its predecessor in a fully automated workflow. Determining the exact number of overlapping lipids based on the area occupied by the protein and restricting expansion, compression and energy minimization steps to a subset of relevant lipids through automatically calculated and system-optimized operation parameters, InflateGRO2 yields optimal lipid packing and reduces lipid vacuum exposure to a minimum preserving as much of the equilibrated membrane structure as possible. Applicable to atomistic and coarse grain structures in MARTINI format, InflateGRO2 offers high accuracy, fast performance, and increased application flexibility permitting the easy preparation of systems exhibiting heterogeneous lipid composition as well as embedding proteins into multiple membranes. Both tools can be used separately, in combination with other methods, or in tandem permitting a fully automated workflow while retaining a maximum level of usage control and flexibility. To assess the performance of both methods, we carried out test runs using 22 membrane proteins of different size and transmembrane structure.
Aligning Biomolecular Networks Using Modular Graph Kernels

NASA Astrophysics Data System (ADS)

Towfic, Fadi; Greenlee, M. Heather West; Honavar, Vasant

Comparative analysis of biomolecular networks constructed using measurements from different conditions, tissues, and organisms offer a powerful approach to understanding the structure, function, dynamics, and evolution of complex biological systems. We explore a class of algorithms for aligning large biomolecular networks by breaking down such networks into subgraphs and computing the alignment of the networks based on the alignment of their subgraphs. The resulting subnetworks are compared using graph kernels as scoring functions. We provide implementations of the resulting algorithms as part of BiNA, an open source biomolecular network alignment toolkit. Our experiments using Drosophila melanogaster, Saccharomyces cerevisiae, Mus musculus and Homo sapiens protein-protein interaction networks extracted from the DIP repository of protein-protein interaction data demonstrate that the performance of the proposed algorithms (as measured by % GO term enrichment of subnetworks identified by the alignment) is competitive with some of the state-of-the-art algorithms for pair-wise alignment of large protein-protein interaction networks. Our results also show that the inter-species similarity scores computed based on graph kernels can be used to cluster the species into a species tree that is consistent with the known phylogenetic relationships among the species.
Computational Enzyme Design: Advances, hurdles and possible ways forward

PubMed Central

Linder, Mats

2012-01-01

This mini review addresses recent developments in computational enzyme design. Successful protocols as well as known issues and limitations are discussed from an energetic perspective. It will be argued that improved results can be obtained by including a dynamic treatment in the design protocol. Finally, a molecular dynamics-based approach for evaluating and refining computational designs is presented. PMID:24688650
Experimental validation of arthroscopic cartilage stiffness measurement using enzymatically degraded cartilage samples

NASA Astrophysics Data System (ADS)

Lyyra, T.; Arokoski, J. P. A.; Oksala, N.; Vihko, A.; Hyttinen, M.; Jurvelin, J. S.; Kiviranta, I.

1999-02-01

In order to evaluate the ability of the arthroscopic indentation instrument, originally developed for the measurement of cartilage stiffness during arthroscopy, to detect cartilage degeneration, we compared changes in the stiffness with the structural and constitutional alterations induced by enzymes on the tissue in vitro. The culturing of osteochondral plugs on Petri dishes was initiated in Minimum Essential Medium with Earle's salts and the baseline stiffness was measured. Then, the experimental specimens were digested using trypsin for 24 h, chondroitinase ABC or purified collagenase (type VII) for 24 h or 48 h ( n = 8-15 per group). The control specimens were incubated in the medium. After the enzyme digestion, the end-point stiffness was measured and the specimens for the microscopic analyses were processed. The proteoglycan (PG) distribution was analysed using quantitative microspectrophotometry and the quantitative evaluation of the collagen network was made using a computer-based polarized light microscopy analysis. Decrease of cartilage stiffness was found after 24 h trypsin (36%) and 48 h chondroitinase ABC (24%) digestion corresponding to a decrease of up to 80% and up to 30% in the PG content respectively. Decrease of the superficial zone collagen content or arrangement (78%, ) after 48 h collagenase digestion also induced a decrease (30%, ) in cartilage stiffness. We conclude that our instrument is capable of detecting early structural and compositional changes related to cartilage degeneration.
A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function

PubMed Central

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A.

2012-01-01

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that the functional evolution can be inferred from the changes in the protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced Cα representation of the protein structure while enzymatic function is described by Enzyme Commission (EC) numbers. Similarity of the binding pocket dynamics at each branch of the protein family’s phylogeny was analyzed in two ways: 1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and 2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the alpha-amylase, D-isomer specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal modes analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. PMID:22651983
A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function.

PubMed

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A

2012-09-21

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that functional evolution can be inferred from the changes in protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced C(α) representation of the protein structure while enzymatic function is described by Enzyme Commission numbers. Similarity of the binding pocket dynamics at each branch of the protein family's phylogeny was analyzed in two ways: (1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and (2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the α-amylase, D-isomer-specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal mode analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. Copyright © 2012 Elsevier Ltd. All rights reserved.
SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

PubMed Central

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

2013-01-01

Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158
Model-based estimation and control for off-axis parabolic mirror alignment

NASA Astrophysics Data System (ADS)

Fang, Joyce; Savransky, Dmitry

2018-02-01

This paper propose an model-based estimation and control method for an off-axis parabolic mirror (OAP) alignment. Current studies in automated optical alignment systems typically require additional wavefront sensors. We propose a self-aligning method using only focal plane images captured by the existing camera. Image processing methods and Karhunen-Loève (K-L) decomposition are used to extract measurements for the observer in closed-loop control system. Our system has linear dynamic in state transition, and a nonlinear mapping from the state to the measurement. An iterative extended Kalman filter (IEKF) is shown to accurately predict the unknown states, and nonlinear observability is discussed. Linear-quadratic regulator (LQR) is applied to correct the misalignments. The method is validated experimentally on the optical bench with a commercial OAP. We conduct 100 tests in the experiment to demonstrate the consistency in between runs.
Using the small alignment index chaos indicator to characterize the vibrational dynamics of a molecular system: LiNC-LiCN.

PubMed

Benitez, P; Losada, J C; Benito, R M; Borondo, F

2015-10-01

A study of the dynamical characteristics of the phase space corresponding to the vibrations of the LiNC-LiCN molecule using an analysis based on the small alignment index (SALI) is presented. SALI is a good indicator of chaos that can easily determine whether a given trajectory is regular or chaotic regardless of the dimensionality of the system, and can also provide a wealth of dynamical information when conveniently implemented. In two-dimensional (2D) systems SALI maps are computed as 2D phase space representations, where the SALI asymptotic values are represented in color scale. We show here how these maps provide full information on the dynamical phase space structure of the LiNC-LiCN system, even quantifying numerically the volume of the different zones of chaos and regularity as a function of the molecule excitation energy.
2008 Annual Report

DTIC Science & Technology

2008-01-01

sensors. We will engineer a collection of protein-based switches that are capable of dynamically responding to our desired end-product D-BT over a...locations in the cells; (2) enables control over the molecular ratios of pathway enzymes; and (3) minimizes metabolic cross-talk and side reactions by...pathway enzymes into either static or dynamic channels will be performed by: (1) construction of fusion proteins (static); (2) post-translational protein
Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2.

PubMed

Smichi, Nabil; Othman, Houcemeddine; Achouri, Neila; Noiriel, Alexandre; Triki, Soumaya; Arondel, Vincent; Srairi-Abid, Najet; Abousalham, Abdelkarim; Gargouri, Youssef; Miled, Nabil; Fendri, Ahmed

2018-03-01

Here we report the cDNA cloning of a phospholipase A 2 (PLA 2 ) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA 2 . In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA 2 are closer to avian PLA 2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA 2 (AsPLA 2 ). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA 2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg -1 measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA 2 . By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA 2 at high temperatures was explained. Using the monomolecular film technique, AsPLA 2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm -1 . Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA 2 , provides new insights on structure-activity relationships of fish PLA 2 . Copyright © 2017 Elsevier B.V. All rights reserved.
Log-odds sequence logos

PubMed Central

Yu, Yi-Kuo; Capra, John A.; Stojmirović, Aleksandar; Landsman, David; Altschul, Stephen F.

2015-01-01

Motivation: DNA and protein patterns are usefully represented by sequence logos. However, the methods for logo generation in common use lack a proper statistical basis, and are non-optimal for recognizing functionally relevant alignment columns. Results: We redefine the information at a logo position as a per-observation multiple alignment log-odds score. Such scores are positive or negative, depending on whether a column’s observations are better explained as arising from relatedness or chance. Within this framework, we propose distinct normalized maximum likelihood and Bayesian measures of column information. We illustrate these measures on High Mobility Group B (HMGB) box proteins and a dataset of enzyme alignments. Particularly in the context of protein alignments, our measures improve the discrimination of biologically relevant positions. Availability and implementation: Our new measures are implemented in an open-source Web-based logo generation program, which is available at http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/logoddslogo/index.html. A stand-alone version of the program is also available from this site. Contact: altschul@ncbi.nlm.nih.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25294922
A search for sources of drug resistance by the 4D-QSAR analysis of a set of antimalarial dihydrofolate reductase inhibitors

NASA Astrophysics Data System (ADS)

Santos-Filho, Osvaldo Andrade; Hopfinger, Anton J.

2001-01-01

A set of 18 structurally diverse antifolates including pyrimethamine, cycloguanil, methotrexate, aminopterin and trimethoprim, and 13 pyrrolo[2,3-d]pyrimidines were studied using four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis. The corresponding biological activities of these compounds include IC50 inhibition constants for both the wild type, and a specific mutant type of Plasmodium falciparum dihydrofolate reductase (DHFR). Two thousand conformations of each analog were sampled to generate a conformational ensemble profile (CEP) from a molecular dynamics simulation (MDS) of 100,000 conformer trajectory states. Each sampled conformation was placed in a 1 Å cubic grid cell lattice for each of five trial alignments. The frequency of occupation of each grid cell was computed for each of six types of pharmacophore groups of atoms of each compound. These grid cell occupancy descriptors (GCODs) were then used as a descriptor pool to construct 4D-QSAR models. Models for inhibition of both the `wild' type and the mutant enzyme were generated which provide detailed spatial pharmacophore requirements for inhibition in terms of atom types and their corresponding relative locations in space. The 4D-QSAR models indicate some structural features perhaps relevant to the mechanism of resistance of the Plasmodium falciparum DHFR to current antimalarials. One feature identified is a slightly different binding alignment of the ligands to the mutant form of the enzyme as compared to the wild type.
Social dynamics within decomposer communities lead to nitrogen retention and organic matter build-up in soils

PubMed Central

Kaiser, Christina; Franklin, Oskar; Richter, Andreas; Dieckmann, Ulf

2015-01-01

The chemical structure of organic matter has been shown to be only marginally important for its decomposability by microorganisms. The question of why organic matter does accumulate in the face of powerful microbial degraders is thus key for understanding terrestrial carbon and nitrogen cycling. Here we demonstrate, based on an individual-based microbial community model, that social dynamics among microbes producing extracellular enzymes (‘decomposers') and microbes exploiting the catalytic activities of others (‘cheaters') regulate organic matter turnover. We show that the presence of cheaters increases nitrogen retention and organic matter build-up by downregulating the ratio of extracellular enzymes to total microbial biomass, allowing nitrogen-rich microbial necromass to accumulate. Moreover, increasing catalytic efficiencies of enzymes are outbalanced by a strong negative feedback on enzyme producers, leading to less enzymes being produced at the community level. Our results thus reveal a possible control mechanism that may buffer soil CO2 emissions in a future climate. PMID:26621582
Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming.

PubMed

Zhou, Zhanping; Liu, Yang; Chang, Zhenying; Wang, Huilin; Leier, André; Marquez-Lago, Tatiana T; Ma, Yanhe; Li, Jian; Song, Jiangning

2017-04-01

Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are promising, eco-friendly substitutes for conventional chemical degumming processes. However, to potentiate the enzymes' use for industrial applications, resolving the molecular structure to elucidate catalytic mechanisms becomes necessary. In this manuscript, we report the high resolution (1.45 Å) crystal structure of pectate lyase (pelN) from Paenibacillus sp. 0602 in apo form. Through sequence alignment and structural superposition with other members of the polysaccharide lyase (PL) family 1 (PL1), we determined that pelN shares the characteristic right-handed β-helix and is structurally similar to other members of the PL1 family, while exhibiting key differences in terms of catalytic and substrate binding residues. Then, based on information from structure alignments with other PLs, we engineered a novel pelN. Our rational design yielded a pelN mutant with a temperature for enzymatic activity optimally shifted from 67.5 to 60 °C. Most importantly, this pelN mutant displayed both higher specific activity and ramie fiber degumming ability when compared with the wild-type enzyme. Altogether, our rational design method shows great potential for industrial applications. Moreover, we expect the reported high-resolution crystal structure to provide a solid foundation for future rational, structure-based engineering of genetically enhanced pelNs.
Dynamic interferometer alignment and its utility in UV Fourier transform spectrometer systems

NASA Technical Reports Server (NTRS)

Dorval, Rick K.; Engel, James R.; Wyntjes, Geert J.

1993-01-01

Dynamic alignment has been demonstrated as a practical approach to alignment maintenance for systems in the infrared region of the spectrum. On the basis of work done by OPTRA, this technique was introduced in commercial Fourier transform spectrometer systems in 1982 and in various forms is now available from a number of manufacturers. This paper reports on work by OPTRA to extend the basic technique to systems operating in the ultraviolet. In addition, this paper reports the preliminary results of the development of an alignment system using a laser diode in place of a gas laser normally found in dynamic alignment systems. A unique optical system and spatial heterodyne technique allows for achievement of a metrology system with characteristics that fully satisfy the requirements of an ultraviolet spectrometer system.
Solution structural ensembles of substrate-free cytochrome P450(cam).

PubMed

Asciutto, Eliana K; Young, Matthew J; Madura, Jeffry; Pochapsky, Susan Sondej; Pochapsky, Thomas C

2012-04-24

Removal of substrate (+)-camphor from the active site of cytochrome P450(cam) (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The (1)H-(15)N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for (15)N-(1)H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. Nuclear magnetic resonance-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information about relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of the substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form.

Disposable sensor based on enzyme-free Ni nanowire array electrode to detect glutamate.

PubMed

Jamal, Mamun; Hasan, Maksudul; Mathewson, Alan; Razeeb, Kafil M

2013-02-15

Enzyme free electrochemical sensor platform based on a vertically aligned nickel nanowire array (NiNAE) and Pt coated nickel nanowire array (Pt/NiNAE) have been developed to detect glutamate. Morphological characterisation of Ni electrodes was carried out using scanning and transmission electron microscopy combined with energy dispersive X-ray (SEM-EDX), X-ray diffraction (XRD) and transmission electron microscopy (TEM). Cyclic voltammetry (CV) and amperometry were used to evaluate the catalytic activity of the NiNAE and the Pt/NiNAE for glutamate. It has been found that both NiNAE and Pt/NiNAE electrodes showed remarkably enhanced electrocatalytic activity towards glutamate compared to planar Ni electrodes, and showed higher catalytic activity when compared to other metallic nanostructure electrodes such as gold nanowire array electrodes (AuNAE) and Pt coated gold nanowire array electrode (Pt/AuNAE). The sensitivity of NiNAE and Pt/NiNAE has been found to be 65 and 96 μA mM(-1) cm(-2), respectively, which is approximately 6 to 9 times higher than the state of the art glutamate sensor. Under optimal detection conditions, the as prepared sensors exhibited linear behaviour for glutamate detection in the concentration up to 8mM for both NiNAE and Pt/NiNAE with a limit of detection of 68 and 83 μM, respectively. Experimental results show that the vertically aligned ordered nickel nanowire array electrode (NiNAE) has significant promise for fabricating cost effective, enzyme-less, sensitive, stable and selective sensor platform. Copyright © 2012 Elsevier B.V. All rights reserved.
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
Integrated Smartphone-App-Chip System for On-Site Parts-Per-Billion-Level Colorimetric Quantitation of Aflatoxins.

PubMed

Li, Xiaochun; Yang, Fan; Wong, Jessica X H; Yu, Hua-Zhong

2017-09-05

We demonstrate herein an integrated, smartphone-app-chip (SPAC) system for on-site quantitation of food toxins, as demonstrated with aflatoxin B1 (AFB1), at parts-per-billion (ppb) level in food products. The detection is based on an indirect competitive immunoassay fabricated on a transparent plastic chip with the assistance of a microfluidic channel plate. A 3D-printed optical accessory attached to a smartphone is adapted to align the assay chip and to provide uniform illumination for imaging, with which high-quality images of the assay chip are captured by the smartphone camera and directly processed using a custom-developed Android app. The performance of this smartphone-based detection system was tested using both spiked and moldy corn samples; consistent results with conventional enzyme-linked immunosorbent assay (ELISA) kits were obtained. The achieved detection limit (3 ± 1 ppb, equivalent to μg/kg) and dynamic response range (0.5-250 ppb) meet the requested testing standards set by authorities in China and North America. We envision that the integrated SPAC system promises to be a simple and accurate method of food toxin quantitation, bringing much benefit for rapid on-site screening.
Perception of socket alignment perturbations in amputees with transtibial prostheses.

PubMed

Boone, David A; Kobayashi, Toshiki; Chou, Teri G; Arabian, Adam K; Coleman, Kim L; Orendurff, Michael S; Zhang, Ming

2012-01-01

A person with amputation's subjective perception is the only tool available to describe fit and comfort to a prosthetist. However, few studies have investigated the effect of alignment on this perception. The aim of this article is to determine whether people with amputation could perceive the alignment perturbations of their prostheses and effectively communicate them. A randomized controlled perturbation of angular (3 and 6 degrees) and translational (5 and 10 mm) alignments in the sagittal (flexion, extension, and anterior and posterior translations) and coronal (abduction, adduction, and medial and lateral translations) planes were induced from an aligned condition in 11 subjects with transtibial prostheses. The perception was evaluated when standing (static) and immediately after walking (dynamic) using software that used a visual analog scale under each alignment condition. In the coronal plane, Friedman test demonstrated general statistical differences in static (p < 0.001) and dynamic (p < 0.001) measures of perceptions with angular perturbations. In the sagittal plane, it also demonstrated general statistical differences in late-stance dynamic measures of perceptions (p < 0.001) with angular perturbations, as well as in early-stance dynamic measures of perceptions (p < 0.05) with translational perturbations. Fisher exact test suggested that people with amputation's perceptions were good indicators for coronal angle malalignments but less reliable when defining other alignment conditions.
Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

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

Zhang, R.; Evans, G.; Rotella, F.

1999-06-01

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show bio- chemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification ofmore » these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.« less
Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

PubMed Central

Dorner, Mariah E; McMunn, Ryan D; Bartholow, Thomas G; Calhoon, Brecken E; Conlon, Michelle R; Dulli, Jessica M; Fehling, Samuel C; Fisher, Cody R; Hodgson, Shane W; Keenan, Shawn W; Kruger, Alyssa N; Mabin, Justin W; Mazula, Daniel L; Monte, Christopher A; Olthafer, Augustus; Sexton, Ashley E; Soderholm, Beatrice R; Strom, Alexander M; Hati, Sanchita

2015-01-01

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes. PMID:26130403
Optical mounts for harsh environments

NASA Astrophysics Data System (ADS)

Mimovich, Mark E.; Griffee, Jonathan C.; Goodding, James C.

2009-08-01

Development and testing of a lightweight-kinematic optical mount with integrated passive vibration-and-shock mitigation technologies and simple / robust optical alignment functionality is presented. Traditionally, optical mounts are designed for use in laboratory environments where the thermal-mechanical environments are carefully controlled to preserve beam path conditions and background disturbances are minimized to facilitate precise optically based measurements. Today's weapon and surveillance systems, however, have optical sensor suites where static and dynamic alignment performance in the presence of harsh operating environments is required to nearly the same precision and where the system cannot afford the mass of laboratory-grade stabilized mounting systems. Jitter and alignment stability is particularly challenging for larger optics operating within moving vehicles and aircraft where high shock and significant temperature excursions occur. The design intent is to have the mount be suitable for integration into existing defense and security optical systems while also targeting new commercial and military components for improved structural dynamic and thermal distortion performance. A mount suitable for moderate-sized optics and an integrated disturbance-optical metrology system are described. The mount design has performance enhancements derived from the integration of proven aerospace mechanical vibration and shock mitigation technologies (i.e. multi-axis passive isolation and integral damping), precision alignment adjustment and lock-out functionality, high dimensional stability materials and design practices which provide benign optical surface figure errors under harsh thermal-mechanical loading. Optical jitter, alignment, and wave-front performance testing of an eight-inch-aperture optical mount based on this design approach are presented to validate predicted performance improvements over an existing commercial off-the-shelf (COTS) design.
High dynamic range image acquisition based on multiplex cameras

NASA Astrophysics Data System (ADS)

Zeng, Hairui; Sun, Huayan; Zhang, Tinghua

2018-03-01

High dynamic image is an important technology of photoelectric information acquisition, providing higher dynamic range and more image details, and it can better reflect the real environment, light and color information. Currently, the method of high dynamic range image synthesis based on different exposure image sequences cannot adapt to the dynamic scene. It fails to overcome the effects of moving targets, resulting in the phenomenon of ghost. Therefore, a new high dynamic range image acquisition method based on multiplex cameras system was proposed. Firstly, different exposure images sequences were captured with the camera array, using the method of derivative optical flow based on color gradient to get the deviation between images, and aligned the images. Then, the high dynamic range image fusion weighting function was established by combination of inverse camera response function and deviation between images, and was applied to generated a high dynamic range image. The experiments show that the proposed method can effectively obtain high dynamic images in dynamic scene, and achieves good results.
Using Variable-Length Aligned Fragment Pairs and an Improved Transition Function for Flexible Protein Structure Alignment.

PubMed

Cao, Hu; Lu, Yonggang

2017-01-01

With the rapid growth of known protein 3D structures in number, how to efficiently compare protein structures becomes an essential and challenging problem in computational structural biology. At present, many protein structure alignment methods have been developed. Among all these methods, flexible structure alignment methods are shown to be superior to rigid structure alignment methods in identifying structure similarities between proteins, which have gone through conformational changes. It is also found that the methods based on aligned fragment pairs (AFPs) have a special advantage over other approaches in balancing global structure similarities and local structure similarities. Accordingly, we propose a new flexible protein structure alignment method based on variable-length AFPs. Compared with other methods, the proposed method possesses three main advantages. First, it is based on variable-length AFPs. The length of each AFP is separately determined to maximally represent a local similar structure fragment, which reduces the number of AFPs. Second, it uses local coordinate systems, which simplify the computation at each step of the expansion of AFPs during the AFP identification. Third, it decreases the number of twists by rewarding the situation where nonconsecutive AFPs share the same transformation in the alignment, which is realized by dynamic programming with an improved transition function. The experimental data show that compared with FlexProt, FATCAT, and FlexSnap, the proposed method can achieve comparable results by introducing fewer twists. Meanwhile, it can generate results similar to those of the FATCAT method in much less running time due to the reduced number of AFPs.
Enhanced spatio-temporal alignment of plantar pressure image sequences using B-splines.

PubMed

Oliveira, Francisco P M; Tavares, João Manuel R S

2013-03-01

This article presents an enhanced methodology to align plantar pressure image sequences simultaneously in time and space. The temporal alignment of the sequences is accomplished using B-splines in the time modeling, and the spatial alignment can be attained using several geometric transformation models. The methodology was tested on a dataset of 156 real plantar pressure image sequences (3 sequences for each foot of the 26 subjects) that was acquired using a common commercial plate during barefoot walking. In the alignment of image sequences that were synthetically deformed both in time and space, an outstanding accuracy was achieved with the cubic B-splines. This accuracy was significantly better (p < 0.001) than the one obtained using the best solution proposed in our previous work. When applied to align real image sequences with unknown transformation involved, the alignment based on cubic B-splines also achieved superior results than our previous methodology (p < 0.001). The consequences of the temporal alignment on the dynamic center of pressure (COP) displacement was also assessed by computing the intraclass correlation coefficients (ICC) before and after the temporal alignment of the three image sequence trials of each foot of the associated subject at six time instants. The results showed that, generally, the ICCs related to the medio-lateral COP displacement were greater when the sequences were temporally aligned than the ICCs of the original sequences. Based on the experimental findings, one can conclude that the cubic B-splines are a remarkable solution for the temporal alignment of plantar pressure image sequences. These findings also show that the temporal alignment can increase the consistency of the COP displacement on related acquired plantar pressure image sequences.
Note: A simple image processing based fiducial auto-alignment method for sample registration.

PubMed

Robertson, Wesley D; Porto, Lucas R; Ip, Candice J X; Nantel, Megan K T; Tellkamp, Friedjof; Lu, Yinfei; Miller, R J Dwayne

2015-08-01

A simple method for the location and auto-alignment of sample fiducials for sample registration using widely available MATLAB/LabVIEW software is demonstrated. The method is robust, easily implemented, and applicable to a wide variety of experiment types for improved reproducibility and increased setup speed. The software uses image processing to locate and measure the diameter and center point of circular fiducials for distance self-calibration and iterative alignment and can be used with most imaging systems. The method is demonstrated to be fast and reliable in locating and aligning sample fiducials, provided here by a nanofabricated array, with accuracy within the optical resolution of the imaging system. The software was further demonstrated to register, load, and sample the dynamically wetted array.
Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase.

PubMed

Mittal, Monica; Hasan, Mahmudul; Balagunaseelan, Navisraj; Fauland, Alexander; Wheelock, Craig; Rådmark, Olof; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

2017-08-01

A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. Here we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca 2+ -dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. The study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general. Copyright © 2017 Elsevier B.V. All rights reserved.
Computational Functional Analysis of Lipid Metabolic Enzymes.

PubMed

Bagnato, Carolina; Have, Arjen Ten; Prados, María B; Beligni, María V

2017-01-01

The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.
Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

PubMed

Chen, Hui; Huang, Rui; Zhang, Y-H Percival

2017-06-01

The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).
Effects of geometry and cell-matrix interactions on the mechanics of 3D engineered microtissues

NASA Astrophysics Data System (ADS)

Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

Approaches to measure and control cell-extracellular matrix (ECM) interactions in a dynamic mechanical environment are important both for studies of mechanobiology and for tissue design for bioengineering applications. We have developed a microtissue-based platform capable of controlling the ECM alignment of 3D engineered microtissues while simultaneously permitting measurement of cellular contractile forces and the tissues' mechanical properties. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of flexible elastic pillars. Tissue geometry and ECM alignment are controlled by the pillars' number, shape and location. Optical tracking of the pillars provides readout of the tissues' contractile forces. Magnetic materials bound to selected pillars allow quasi-static or dynamic stretching of the tissue, and together with simultaneous measurements of the tissues' local dynamic strain field, enable characterization of the mechanical properties of the system, including their degree of anisotropy. Results on the effects of symmetry and degree of ECM alignment and organization on the role of cell-ECM interactions in determining tissue mechanical properties will be discussed. This work is supported by NSF CMMI-1463011 and CMMI-1462710.
Theory of the deformation of aligned polyethylene.

PubMed

Hammad, A; Swinburne, T D; Hasan, H; Del Rosso, S; Iannucci, L; Sutton, A P

2015-08-08

Solitons are proposed as the agents of plastic and viscoelastic deformation in aligned polyethylene. Interactions between straight, parallel molecules are mapped rigorously onto the Frenkel-Kontorova model. It is shown that these molecular interactions distribute an applied load between molecules, with a characteristic transfer length equal to the soliton width. Load transfer leads to the introduction of tensile and compressive solitons at the chain ends to mark the onset of plasticity at a well-defined yield stress, which is much less than the theoretical pull-out stress. Interaction energies between solitons and an equation of motion for solitons are derived. The equation of motion is based on Langevin dynamics and the fluctuation-dissipation theorem and it leads to the rigorous definition of an effective mass for solitons. It forms the basis of a soliton dynamics in direct analogy to dislocation dynamics. Close parallels are drawn between solitons in aligned polymers and dislocations in crystals, including the configurational force on a soliton. The origins of the strain rate and temperature dependencies of the viscoelastic behaviour are discussed in terms of the formation energy of solitons. A failure mechanism is proposed involving soliton condensation under a tensile load.
A pharmacophore model specific to active site of CYP1A2 with a novel molecular modeling explorer and CoMFA.

PubMed

Zhang, Tao; Wei, Dong-Qing; Chou, Kuo-Chen

2012-03-01

Comparative molecular field analysis (CoMFA) is a widely used 3D-QSAR method by which we can investigate the potential relation between biological activity of compounds and their structural features. In this study, a new application of this approach is presented by combining the molecular modeling with a new developed pharmacophore model specific to CYP1A2 active site. During constructing the model, we used the molecular dynamics simulation and molecular docking method to select the sensible binding conformations for 17 CYP1A2 substrates based on the experimental data. Subsequently, the results obtained via the alignment of binding conformations of substrates were projected onto the active- site residues, upon which a simple blueprint of active site was produced. It was validated by the experimental and computational results that the model did exhibit the high degree of rationality and provide useful insights into the substrate binding. It is anticipated that our approach can be extended to investigate the protein-ligand interactions for many other enzyme-catalyzed systems as well.
Flexible Semitransparent Energy Harvester with High Pressure Sensitivity and Power Density Based on Laterally Aligned PZT Single-Crystal Nanowires.

PubMed

Zhao, Quan-Liang; He, Guang-Ping; Di, Jie-Jian; Song, Wei-Li; Hou, Zhi-Ling; Tan, Pei-Pei; Wang, Da-Wei; Cao, Mao-Sheng

2017-07-26

A flexible semitransparent energy harvester is assembled based on laterally aligned Pb(Zr 0.52 Ti 0.48 )O 3 (PZT) single-crystal nanowires (NWs). Such a harvester presents the highest open-circuit voltage and a stable area power density of up to 10 V and 0.27 μW/cm 2 , respectively. A high pressure sensitivity of 0.14 V/kPa is obtained in the dynamic pressure sensing, much larger than the values reported in other energy harvesters based on piezoelectric single-crystal NWs. Furthermore, theoretical and finite element analyses also confirm that the piezoelectric voltage constant g 33 of PZT NWs is competitive to the lead-based bulk single crystals and ceramics, and the enhanced pressure sensitivity and power density are substantially linked to the flexible structure with laterally aligned PZT NWs. The energy harvester in this work holds great potential in flexible and transparent sensing and self-powered systems.
Intrinsic fluorescence spectroscopy of glutamate dehydrogenase: Integrated behavior and deconvolution analysis

NASA Astrophysics Data System (ADS)

Pompa, P. P.; Cingolani, R.; Rinaldi, R.

2003-07-01

In this paper, we present a deconvolution method aimed at spectrally resolving the broad fluorescence spectra of proteins, namely, of the enzyme bovine liver glutamate dehydrogenase (GDH). The analytical procedure is based on the deconvolution of the emission spectra into three distinct Gaussian fluorescing bands Gj. The relative changes of the Gj parameters are directly related to the conformational changes of the enzyme, and provide interesting information about the fluorescence dynamics of the individual emitting contributions. Our deconvolution method results in an excellent fitting of all the spectra obtained with GDH in a number of experimental conditions (various conformational states of the protein) and describes very well the dynamics of a variety of phenomena, such as the dependence of hexamers association on protein concentration, the dynamics of thermal denaturation, and the interaction process between the enzyme and external quenchers. The investigation was carried out by means of different optical experiments, i.e., native enzyme fluorescence, thermal-induced unfolding, and fluorescence quenching studies, utilizing both the analysis of the “average” behavior of the enzyme and the proposed deconvolution approach.
A polarized view on DNA under tension

NASA Astrophysics Data System (ADS)

van Mameren, Joost; Vermeulen, Karen; Wuite, Gijs J. L.; Peterman, Erwin J. G.

2018-03-01

In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of double-stranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

PubMed

Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

2016-03-28

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.
Robust video copy detection approach based on local tangent space alignment

NASA Astrophysics Data System (ADS)

Nie, Xiushan; Qiao, Qianping

2012-04-01

We propose a robust content-based video copy detection approach based on local tangent space alignment (LTSA), which is an efficient dimensionality reduction algorithm. The idea is motivated by the fact that the content of video becomes richer and the dimension of content becomes higher. It does not give natural tools for video analysis and understanding because of the high dimensionality. The proposed approach reduces the dimensionality of video content using LTSA, and then generates video fingerprints in low dimensional space for video copy detection. Furthermore, a dynamic sliding window is applied to fingerprint matching. Experimental results show that the video copy detection approach has good robustness and discrimination.
The dynamic nature of alignment and variations in normal knees.

PubMed

Deep, K; Eachempati, K K; Apsingi, S

2015-04-01

The restoration of knee alignment is an important goal during total knee arthroplasty (TKA). In the past surgeons aimed to restore neutral limb alignment during surgery. However, previous studies have demonstrated alignment to be dynamic, varying depending on the position of the limb and the degree of weight-bearing, and between patients. We used a validated computer navigation system to measure the femorotibial mechanical angle (FTMA) in 264 knees in 77 male and 55 female healthy volunteers aged 18 to 35 years (mean 26.2). We found the mean supine alignment to be a varus angle of 1.2° (standard deviation (sd) 4), with few patients having neutral alignment. FTMA differs significantly between males and females (with a mean varus of 1.7° (sd 4) and 0.4° (sd 3.9), respectively; p = 0.008). It changes significantly with posture, the knee hyperextending by a mean of 5.6°, and coronal plane alignment becoming more varus by 2.2° (sd 3.6) on standing compared with supine. Knee alignment is different in different individuals and is dynamic in nature, changing with different postures. This may have implications for the assessment of alignment in TKA, which is achieved in non-weight-bearing conditions and which may not represent the situation observed during weight-bearing. ©2015 The British Editorial Society of Bone & Joint Surgery.
A hybrid short read mapping accelerator

PubMed Central

2013-01-01

Background The rapid growth of short read datasets poses a new challenge to the short read mapping problem in terms of sensitivity and execution speed. Existing methods often use a restrictive error model for computing the alignments to improve speed, whereas more flexible error models are generally too slow for large-scale applications. A number of short read mapping software tools have been proposed. However, designs based on hardware are relatively rare. Field programmable gate arrays (FPGAs) have been successfully used in a number of specific application areas, such as the DSP and communications domains due to their outstanding parallel data processing capabilities, making them a competitive platform to solve problems that are “inherently parallel”. Results We present a hybrid system for short read mapping utilizing both FPGA-based hardware and CPU-based software. The computation intensive alignment and the seed generation operations are mapped onto an FPGA. We present a computationally efficient, parallel block-wise alignment structure (Align Core) to approximate the conventional dynamic programming algorithm. The performance is compared to the multi-threaded CPU-based GASSST and BWA software implementations. For single-end alignment, our hybrid system achieves faster processing speed than GASSST (with a similar sensitivity) and BWA (with a higher sensitivity); for pair-end alignment, our design achieves a slightly worse sensitivity than that of BWA but has a higher processing speed. Conclusions This paper shows that our hybrid system can effectively accelerate the mapping of short reads to a reference genome based on the seed-and-extend approach. The performance comparison to the GASSST and BWA software implementations under different conditions shows that our hybrid design achieves a high degree of sensitivity and requires less overall execution time with only modest FPGA resource utilization. Our hybrid system design also shows that the performance bottleneck for the short read mapping problem can be changed from the alignment stage to the seed generation stage, which provides an additional requirement for the future development of short read aligners. PMID:23441908
Mechanism for Collective Cell Alignment in Myxococcus xanthus Bacteria

PubMed Central

Balagam, Rajesh; Igoshin, Oleg A.

2015-01-01

Myxococcus xanthus cells self-organize into aligned groups, clusters, at various stages of their lifecycle. Formation of these clusters is crucial for the complex dynamic multi-cellular behavior of these bacteria. However, the mechanism underlying the cell alignment and clustering is not fully understood. Motivated by studies of clustering in self-propelled rods, we hypothesized that M. xanthus cells can align and form clusters through pure mechanical interactions among cells and between cells and substrate. We test this hypothesis using an agent-based simulation framework in which each agent is based on the biophysical model of an individual M. xanthus cell. We show that model agents, under realistic cell flexibility values, can align and form cell clusters but only when periodic reversals of cell directions are suppressed. However, by extending our model to introduce the observed ability of cells to deposit and follow slime trails, we show that effective trail-following leads to clusters in reversing cells. Furthermore, we conclude that mechanical cell alignment combined with slime-trail-following is sufficient to explain the distinct clustering behaviors observed for wild-type and non-reversing M. xanthus mutants in recent experiments. Our results are robust to variation in model parameters, match the experimentally observed trends and can be applied to understand surface motility patterns of other bacterial species. PMID:26308508
Analytical structure, dynamics, and coarse graining of a kinetic model of an active fluid

NASA Astrophysics Data System (ADS)

Gao, Tong; Betterton, Meredith D.; Jhang, An-Sheng; Shelley, Michael J.

2017-09-01

We analyze one of the simplest active suspensions with complex dynamics: a suspension of immotile "extensor" particles that exert active extensile dipolar stresses on the fluid in which they are immersed. This is relevant to several experimental systems, such as recently studied tripartite rods that create extensile flows by consuming a chemical fuel. We first describe the system through a Doi-Onsager kinetic theory based on microscopic modeling. This theory captures the active stresses produced by the particles that can drive hydrodynamic instabilities, as well as the steric interactions of rodlike particles that lead to nematic alignment. This active nematic system yields complex flows and disclination defect dynamics very similar to phenomenological Landau-deGennes Q -tensor theories for active nematic fluids, as well as by more complex Doi-Onsager theories for polar microtubule-motor-protein systems. We apply the quasiequilibrium Bingham closure, used to study suspensions of passive microscopic rods, to develop a nonstandard Q -tensor theory. We demonstrate through simulation that this B Q -tensor theory gives an excellent analytical and statistical accounting of the suspension's complex dynamics, at a far reduced computational cost. Finally, we apply the B Q -tensor model to study the dynamics of extensor suspensions in circular and biconcave domains. In circular domains, we reproduce previous results for systems with weak nematic alignment, but for strong alignment we find unusual dynamics with activity-controlled defect production and absorption at the boundaries of the domain. In biconcave domains, a Fredericks-like transition occurs as the width of the neck connecting the two disks is varied.
BigFoot: Bayesian alignment and phylogenetic footprinting with MCMC

PubMed Central

Satija, Rahul; Novák, Ádám; Miklós, István; Lyngsø, Rune; Hein, Jotun

2009-01-01

Background We have previously combined statistical alignment and phylogenetic footprinting to detect conserved functional elements without assuming a fixed alignment. Considering a probability-weighted distribution of alignments removes sensitivity to alignment errors, properly accommodates regions of alignment uncertainty, and increases the accuracy of functional element prediction. Our method utilized standard dynamic programming hidden markov model algorithms to analyze up to four sequences. Results We present a novel approach, implemented in the software package BigFoot, for performing phylogenetic footprinting on greater numbers of sequences. We have developed a Markov chain Monte Carlo (MCMC) approach which samples both sequence alignments and locations of slowly evolving regions. We implement our method as an extension of the existing StatAlign software package and test it on well-annotated regions controlling the expression of the even-skipped gene in Drosophila and the α-globin gene in vertebrates. The results exhibit how adding additional sequences to the analysis has the potential to improve the accuracy of functional predictions, and demonstrate how BigFoot outperforms existing alignment-based phylogenetic footprinting techniques. Conclusion BigFoot extends a combined alignment and phylogenetic footprinting approach to analyze larger amounts of sequence data using MCMC. Our approach is robust to alignment error and uncertainty and can be applied to a variety of biological datasets. The source code and documentation are publicly available for download from PMID:19715598
BigFoot: Bayesian alignment and phylogenetic footprinting with MCMC.

PubMed

Satija, Rahul; Novák, Adám; Miklós, István; Lyngsø, Rune; Hein, Jotun

2009-08-28

We have previously combined statistical alignment and phylogenetic footprinting to detect conserved functional elements without assuming a fixed alignment. Considering a probability-weighted distribution of alignments removes sensitivity to alignment errors, properly accommodates regions of alignment uncertainty, and increases the accuracy of functional element prediction. Our method utilized standard dynamic programming hidden markov model algorithms to analyze up to four sequences. We present a novel approach, implemented in the software package BigFoot, for performing phylogenetic footprinting on greater numbers of sequences. We have developed a Markov chain Monte Carlo (MCMC) approach which samples both sequence alignments and locations of slowly evolving regions. We implement our method as an extension of the existing StatAlign software package and test it on well-annotated regions controlling the expression of the even-skipped gene in Drosophila and the alpha-globin gene in vertebrates. The results exhibit how adding additional sequences to the analysis has the potential to improve the accuracy of functional predictions, and demonstrate how BigFoot outperforms existing alignment-based phylogenetic footprinting techniques. BigFoot extends a combined alignment and phylogenetic footprinting approach to analyze larger amounts of sequence data using MCMC. Our approach is robust to alignment error and uncertainty and can be applied to a variety of biological datasets. The source code and documentation are publicly available for download from http://www.stats.ox.ac.uk/~satija/BigFoot/
Predicting Catalytic Proton Donors and Nucleophiles in Enzymes: How Adding Dynamics Helps Elucidate the Structure-Function Relationships.

PubMed

Huang, Yandong; Yue, Zhi; Tsai, Cheng-Chieh; Henderson, Jack A; Shen, Jana

2018-03-15

Despite the relevance of understanding structure-function relationships, robust prediction of proton donors and nucleophiles in enzyme active sites remains challenging. Here we tested three types of state-of-the-art computational methods to calculate the p K a 's of the buried and hydrogen bonded catalytic dyads in five enzymes. We asked the question what determines the p K a order, i.e., what makes a residue proton donor vs a nucleophile. The continuous constant pH molecular dynamics simulations captured the experimental p K a orders and revealed that the negative nucleophile is stabilized by increased hydrogen bonding and solvent exposure as compared to the proton donor. Surprisingly, this simple trend is not apparent from crystal structures and the static structure-based calculations. While the generality of the findings awaits further testing via a larger set of data, they underscore the role of dynamics in bridging enzyme structures and functions.
Profile-Based LC-MS Data Alignment—A Bayesian Approach

PubMed Central

Tsai, Tsung-Heng; Tadesse, Mahlet G.; Wang, Yue; Ressom, Habtom W.

2014-01-01

A Bayesian alignment model (BAM) is proposed for alignment of liquid chromatography-mass spectrometry (LC-MS) data. BAM belongs to the category of profile-based approaches, which are composed of two major components: a prototype function and a set of mapping functions. Appropriate estimation of these functions is crucial for good alignment results. BAM uses Markov chain Monte Carlo (MCMC) methods to draw inference on the model parameters and improves on existing MCMC-based alignment methods through 1) the implementation of an efficient MCMC sampler and 2) an adaptive selection of knots. A block Metropolis-Hastings algorithm that mitigates the problem of the MCMC sampler getting stuck at local modes of the posterior distribution is used for the update of the mapping function coefficients. In addition, a stochastic search variable selection (SSVS) methodology is used to determine the number and positions of knots. We applied BAM to a simulated data set, an LC-MS proteomic data set, and two LC-MS metabolomic data sets, and compared its performance with the Bayesian hierarchical curve registration (BHCR) model, the dynamic time-warping (DTW) model, and the continuous profile model (CPM). The advantage of applying appropriate profile-based retention time correction prior to performing a feature-based approach is also demonstrated through the metabolomic data sets. PMID:23929872
In-flight alignment using H ∞ filter for strapdown INS on aircraft.

PubMed

Pei, Fu-Jun; Liu, Xuan; Zhu, Li

2014-01-01

In-flight alignment is an effective way to improve the accuracy and speed of initial alignment for strapdown inertial navigation system (INS). During the aircraft flight, strapdown INS alignment was disturbed by lineal and angular movements of the aircraft. To deal with the disturbances in dynamic initial alignment, a novel alignment method for SINS is investigated in this paper. In this method, an initial alignment error model of SINS in the inertial frame is established. The observability of the system is discussed by piece-wise constant system (PWCS) theory and observable degree is computed by the singular value decomposition (SVD) theory. It is demonstrated that the system is completely observable, and all the system state parameters can be estimated by optimal filter. Then a H ∞ filter was designed to resolve the uncertainty of measurement noise. The simulation results demonstrate that the proposed algorithm can reach a better accuracy under the dynamic disturbance condition.
Molecular alignment and orientation with a hybrid Raman scattering technique

NASA Astrophysics Data System (ADS)

Bustard, Philip J.; Lausten, R.; Sussman, Benjamin J.

2012-11-01

We demonstrate a scheme for the preparation of molecular alignment and angular momentum orientation using a hybrid combination of two limits of Raman scattering. First a weak, impulsive pump pulse initializes the system via the nonresonant dynamic Stark effect. Then, having overcome the influence of the vacuum fluctuations, an amplification pulse selectively enhances the initial coherences by transient stimulated Raman scattering, generating alignment and angular momentum orientation of molecular hydrogen. The amplitude and phase of the resulting coherent dynamics are experimentally probed, indicating an amplification factor of 4.5. An analytic theory is developed to model the dynamics.
Modulating RNA Alignment Using Directional Dynamic Kinks: Application in Determining an Atomic-Resolution Ensemble for a Hairpin using NMR Residual Dipolar Couplings.

PubMed

Salmon, Loïc; Giambaşu, George M; Nikolova, Evgenia N; Petzold, Katja; Bhattacharya, Akash; Case, David A; Al-Hashimi, Hashim M

2015-10-14

Approaches that combine experimental data and computational molecular dynamics (MD) to determine atomic resolution ensembles of biomolecules require the measurement of abundant experimental data. NMR residual dipolar couplings (RDCs) carry rich dynamics information, however, difficulties in modulating overall alignment of nucleic acids have limited the ability to fully extract this information. We present a strategy for modulating RNA alignment that is based on introducing variable dynamic kinks in terminal helices. With this strategy, we measured seven sets of RDCs in a cUUCGg apical loop and used this rich data set to test the accuracy of an 0.8 μs MD simulation computed using the Amber ff10 force field as well as to determine an atomic resolution ensemble. The MD-generated ensemble quantitatively reproduces the measured RDCs, but selection of a sub-ensemble was required to satisfy the RDCs within error. The largest discrepancies between the RDC-selected and MD-generated ensembles are observed for the most flexible loop residues and backbone angles connecting the loop to the helix, with the RDC-selected ensemble resulting in more uniform dynamics. Comparison of the RDC-selected ensemble with NMR spin relaxation data suggests that the dynamics occurs on the ps-ns time scales as verified by measurements of R(1ρ) relaxation-dispersion data. The RDC-satisfying ensemble samples many conformations adopted by the hairpin in crystal structures indicating that intrinsic plasticity may play important roles in conformational adaptation. The approach presented here can be applied to test nucleic acid force fields and to characterize dynamics in diverse RNA motifs at atomic resolution.
Quadrupole Alignment and Trajectory Correction for Future Linear Colliders: SLC Tests of a Dispersion-Free Steering Algorithm

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

Assmann, R

2004-06-08

The feasibility of future linear colliders depends on achieving very tight alignment and steering tolerances. All proposals (NLC, JLC, CLIC, TESLA and S-BAND) currently require a total emittance growth in the main linac of less than 30-100% [1]. This should be compared with a 100% emittance growth in the much smaller SLC linac [2]. Major advances in alignment and beam steering techniques beyond those used in the SLC are necessary for the next generation of linear colliders. In this paper, we present an experimental study of quadrupole alignment with a dispersion-free steering algorithm. A closely related method (wakefield-free steering) takesmore » into account wakefield effects [3]. However, this method can not be studied at the SLC. The requirements for future linear colliders lead to new and unconventional ideas about alignment and beam steering. For example, no dipole correctors are foreseen for the standard trajectory correction in the NLC [4]; beam steering will be done by moving the quadrupole positions with magnet movers. This illustrates the close symbiosis between alignment, beam steering and beam dynamics that will emerge. It is no longer possible to consider the accelerator alignment as static with only a few surveys and realignments per year. The alignment in future linear colliders will be a dynamic process in which the whole linac, with thousands of beam-line elements, is aligned in a few hours or minutes, while the required accuracy of about 5 pm for the NLC quadrupole alignment [4] is a factor of 20 higher than in existing accelerators. The major task in alignment and steering is the accurate determination of the optimum beam-line position. Ideally one would like all elements to be aligned along a straight line. However, this is not practical. Instead a ''smooth curve'' is acceptable as long as its wavelength is much longer than the betatron wavelength of the accelerated beam. Conventional alignment methods are limited in accuracy by errors in the survey and the fiducials. Beam-based alignment methods ideally only depend upon the BPM resolution and generally provide much better precision. Many of those techniques are described in other contributions to this workshop. In this paper we describe our experiences with a dispersion-free steering algorithm for linacs. This algorithm was first suggested by Raubenheimer and Ruth in 1990 [5]. It h as been studied in simulations for NLC [5], TESLA [6], the S-BAND proposal [7] and CLIC [8]. The dispersion-free steering technique can be applied to the whole linac at once and returns the alignment (or trajectory) that minimizes the dispersive emittance growth of the beam. Thus it allows an extremely fast alignment of the beam-line. As we will show dispersion-free steering is only sensitive to quadrupole misalignments. Wakefield-free steering [3] as mentioned before is a closely related technique that minimizes the emittance growth caused by both dispersion and wakefields. Due to hardware limitations (i.e. insufficient relative range of power supplies) we could not study this method experimentally in the SLC. However, its systematics are very similar to those of dispersion-free steering. The studies of dispersion-free steering which are presented made extensive use of the unique potential of the SLC as the only operating linear collider. We used it to study the performance and problems of advanced beam-based optimization tools in a real beam-line environment and on a large scale. We should mention that the SLC has utilized beam-based alignment for years [9], using the difference of electron and positron trajectories. This method, however, cannot be used in future linear colliders. The goal of our work is to demonstrate the performance of advanced beam-based alignment techniques in linear colliders and to anticipate possible reality-related problems. Those can then be solved in the design state for the next generation of linear colliders.« less
Probing topology and dynamics of the second transmembrane domain (M2δ) of the acetyl choline receptor using magnetically aligned lipid bilayers (bicelles) and EPR spectroscopy.

PubMed

Sahu, Indra D; Mayo, Daniel J; Subbaraman, Nidhi; Inbaraj, Johnson J; McCarrick, Robert M; Lorigan, Gary A

2017-08-01

Characterizing membrane protein structure and dynamics in the lipid bilayer membrane is very important but experimentally challenging. EPR spectroscopy offers a unique set of techniques to investigate a membrane protein structure, dynamics, topology, and distance constraints in lipid bilayers. Previously our lab demonstrated the use of magnetically aligned phospholipid bilayers (bicelles) for probing topology and dynamics of the membrane peptide M2δ of the acetyl choline receptor (AchR) as a proof of concept. In this study, magnetically aligned phospholipid bilayers and rigid spin labels were further utilized to provide improved dynamic information and topology of M2δ peptide. Seven TOAC-labeled AchR M2δ peptides were synthesized to demonstrate the utility of a multi-labeling amino acid substitution alignment strategy. Our data revealed the helical tilts to be 11°, 17°, 9°, 17°, 16°, 11°, 9°±4° for residues I7TOAC, Q13TOAC, A14TOAC, V15TOAC, C16TOAC, L17TOAC, and L18TOAC, respectively. The average helical tilt of the M2δ peptide was determined to be ∼13°. This study also revealed that the TOAC labels were attached to the M2δ peptide with different dynamics suggesting that the sites towards the C-terminal end are more rigid when compared to the sites towards the N-terminus. The dynamics of the TOAC labeled sites were more resolved in the aligned samples when compared to the randomly disordered samples. This study highlights the use of magnetically aligned lipid bilayer EPR technique to determine a more accurate helical tilt and more resolved local dynamics of AchR M2δ peptide. Copyright © 2017 Elsevier B.V. All rights reserved.
Dissipation dynamics of field-free molecular alignment for symmetric-top molecules: Ethane (C2H6)

NASA Astrophysics Data System (ADS)

Zhang, H.; Billard, F.; Yu, X.; Faucher, O.; Lavorel, B.

2018-03-01

The field-free molecular alignment of symmetric-top molecules, ethane, induced by intense non-resonant linearly polarized femtosecond laser pulses is investigated experimentally in the presence of collisional relaxation. The dissipation dynamics of field-free molecular alignment are measured by the balanced detection of ultrafast molecular birefringence of ethane gas samples at high pressures. By separating the molecular alignment into the permanent alignment and the transient alignment, the decay time-constants of both components are quantified at the same pressure. It is observed that the permanent alignment always decays slower compared to the transient alignment within the measured pressure range. This demonstrates that the propensity of molecules to conserve the orientation of angular momentum during collisions, previously observed for linear species, is also applicable to symmetric-top molecules. The results of this work provide valuable information for further theoretical understanding of collisional relaxation within nonlinear polyatomic molecules, which are expected to present interesting and nontrivial features due to an extra rotational degree of freedom.
Microtubule and Cell Contact Dependency of ER-bound PTP1B Localization in Growth Cones

PubMed Central

Fuentes, Federico

2009-01-01

PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity. PMID:19158394
Alignment dynamics of diffusive scalar gradient in a two-dimensional model flow

NASA Astrophysics Data System (ADS)

Gonzalez, M.

2018-04-01

The Lagrangian two-dimensional approach of scalar gradient kinematics is revisited accounting for molecular diffusion. Numerical simulations are performed in an analytic, parameterized model flow, which enables considering different regimes of scalar gradient dynamics. Attention is especially focused on the influence of molecular diffusion on Lagrangian statistical orientations and on the dynamics of scalar gradient alignment.
First-principles approach to calculating energy level alignment at aqueous semiconductor interfaces.

PubMed

Kharche, Neerav; Muckerman, James T; Hybertsen, Mark S

2014-10-24

A first-principles approach is demonstrated for calculating the relationship between an aqueous semiconductor interface structure and energy level alignment. The physical interface structure is sampled using density functional theory based molecular dynamics, yielding the interface electrostatic dipole. The GW approach from many-body perturbation theory is used to place the electronic band edge energies of the semiconductor relative to the occupied 1b1 energy level in water. The application to the specific cases of nonpolar (101¯0) facets of GaN and ZnO reveals a significant role for the structural motifs at the interface, including the degree of interface water dissociation and the dynamical fluctuations in the interface Zn-O and O-H bond orientations. These effects contribute up to 0.5 eV.
Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

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

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

DOE PAGES

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.; ...

2016-06-01

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less
Enzyme-Regulated Fast Self-Healing of a Pillararene-Based Hydrogel.

PubMed

Zhang, Xin; Xu, Jiayun; Lang, Chao; Qiao, Shanpeng; An, Guo; Fan, Xiaotong; Zhao, Linlu; Hou, Chunxi; Liu, Junqiu

2017-06-12

Self-healing, one of the exciting properties of materials, is frequently used to repair the damage of biological and artificial systems. Here we have used enzymatic catalysis approaches to develop a fast self-healing hydrogel, which has been constructed by dynamic aldimine cross-linking of pillar[5]arene-derivant and dialdehyde-functionalized PEG followed by encapsulation of glucose oxidase (GOx) and catalase (CAT). In specific, the two hydroxyl groups at terminal of PEG 4000 are functionalized with benzaldehydes that can interact with amino-containing pillar[5]arene-derivant through dynamic aldimine cross-links, resulting in reversible dynamic hydrogels. Modulus analysis indicated that storage modulus (G') and loss modulus (G″) of the hydrogel increased obviously as the concentration of dialdehyde-functionalized PEG 4000 (DF-PEG 4000 ) increased or the pH values decreased. Once glucose oxidase (GOx) and catalase (CAT) are located, the hydrogel could be fast repaired, with self-healing efficiency up to 100%. Notably tensile test showed that the repair process of pillararene-based hydrogel can finish in several minutes upon enzyme catalysis, while it needed more than 24 h to achieve this recovery without enzymes. This enzyme-regulated self-healing hydrogel would hold promise for delivering drugs and for soft tissue regeneration in the future.
libgapmis: extending short-read alignments

PubMed Central

2013-01-01

Background A wide variety of short-read alignment programmes have been published recently to tackle the problem of mapping millions of short reads to a reference genome, focusing on different aspects of the procedure such as time and memory efficiency, sensitivity, and accuracy. These tools allow for a small number of mismatches in the alignment; however, their ability to allow for gaps varies greatly, with many performing poorly or not allowing them at all. The seed-and-extend strategy is applied in most short-read alignment programmes. After aligning a substring of the reference sequence against the high-quality prefix of a short read--the seed--an important problem is to find the best possible alignment between a substring of the reference sequence succeeding and the remaining suffix of low quality of the read--extend. The fact that the reads are rather short and that the gap occurrence frequency observed in various studies is rather low suggest that aligning (parts of) those reads with a single gap is in fact desirable. Results In this article, we present libgapmis, a library for extending pairwise short-read alignments. Apart from the standard CPU version, it includes ultrafast SSE- and GPU-based implementations. libgapmis is based on an algorithm computing a modified version of the traditional dynamic-programming matrix for sequence alignment. Extensive experimental results demonstrate that the functions of the CPU version provided in this library accelerate the computations by a factor of 20 compared to other programmes. The analogous SSE- and GPU-based implementations accelerate the computations by a factor of 6 and 11, respectively, compared to the CPU version. The library also provides the user the flexibility to split the read into fragments, based on the observed gap occurrence frequency and the length of the read, thereby allowing for a variable, but bounded, number of gaps in the alignment. Conclusions We present libgapmis, a library for extending pairwise short-read alignments. We show that libgapmis is better-suited and more efficient than existing algorithms for this task. The importance of our contribution is underlined by the fact that the provided functions may be seamlessly integrated into any short-read alignment pipeline. The open-source code of libgapmis is available at http://www.exelixis-lab.org/gapmis. PMID:24564250
Loop conformation and dynamics of the Escherichia coli HPPK apo-enzyme and its binary complex with MgATP.

PubMed

Yang, Rong; Lee, Matthew C; Yan, Honggao; Duan, Yong

2005-07-01

Comparison of the crystallographic and NMR structures of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) suggests that the enzyme may undergo significant conformational change upon binding to its first substrate, ATP. Two of the three surface loops (loop 2 and loop 3) accounting for most of the conformational differences appear to be confined by crystal contacts, raising questions about the putative large-scale induced-fit conformational change of HPPK and the functional roles of the conserved side-chain residues on the loops. To investigate the loop dynamics in crystal-free environment, we carried out molecular dynamics and locally enhanced sampling simulations of the apo-enzyme and the HPPK.MgATP complex. Our simulations showed that the crystallographic B-factors underestimated the loop dynamics considerably. We found that the open-conformation of loop 3 in the binary complex is accessible to the apo-enzyme and is the favored conformation in solution phase. These results revise our previous view of HPPK-substrate interactions and the associated functional mechanism of conformational change. The lessons learned here offer valuable structural insights into the workings of HPPK and should be useful for structure-based drug design.
European Scientific Notes. Volume 37, Number 1.

DTIC Science & Technology

1983-01-31

instantoneous sea-state condition can be tions vary widely in their realism , with computed from a special data base coded some producing dynamic color pictures...between the variables of accuracy, approach channels, the alignment of practicality, realism , and expense. jetties, and the establishment of Because the...tidal current variables The system certainly seems to be valid, have been played into some of the and the smooth dynamics, realism , and simulator runs
Mirroring and beyond: coupled dynamics as a generalized framework for modelling social interactions

PubMed Central

Hasson, Uri; Frith, Chris D.

2016-01-01

When people observe one another, behavioural alignment can be detected at many levels, from the physical to the mental. Likewise, when people process the same highly complex stimulus sequences, such as films and stories, alignment is detected in the elicited brain activity. In early sensory areas, shared neural patterns are coupled to the low-level properties of the stimulus (shape, motion, volume, etc.), while in high-order brain areas, shared neural patterns are coupled to high-levels aspects of the stimulus, such as meaning. Successful social interactions require such alignments (both behavioural and neural), as communication cannot occur without shared understanding. However, we need to go beyond simple, symmetric (mirror) alignment once we start interacting. Interactions are dynamic processes, which involve continuous mutual adaptation, development of complementary behaviour and division of labour such as leader–follower roles. Here, we argue that interacting individuals are dynamically coupled rather than simply aligned. This broader framework for understanding interactions can encompass both processes by which behaviour and brain activity mirror each other (neural alignment), and situations in which behaviour and brain activity in one participant are coupled (but not mirrored) to the dynamics in the other participant. To apply these more sophisticated accounts of social interactions to the study of the underlying neural processes we need to develop new experimental paradigms and novel methods of data analysis PMID:27069044
Grasping Beer Mugs: On the Dynamics of Alignment Effects Induced by Handled Objects

ERIC Educational Resources Information Center

Bub, Daniel N.; Masson, Michael E. J.

2010-01-01

We examined automatic spatial alignment effects evoked by handled objects. Using color as the relevant cue carried by an irrelevant handled object aligned or misaligned with the response hand, responses to color were faster when the handle aligned with the response hand. Alignment effects were observed only when the task was to make a reach and…
Stability limits and dynamics of nonaxisymmetric liquid bridges

NASA Technical Reports Server (NTRS)

Alexander, J. Iwan D.; Resnik, Andy; Kaukler, William F.

1993-01-01

This program of theoretical and experimental ground-based and low gravity research is focussed on the understanding of the dynamics and stability limits of nonaxisymmetric liquid bridges. There are three basic objectives to the proposed work: (1) to determine the stability limits of nonaxisymmetric liquid bridges held between non-coaxially aligned disks; (2) to examine the dynamics of nonaxisymmetric bridges and nonaxisymmetric oscillations of initially axisymmetric bridges (some of these experiments require a low gravity environment and the ground-based research will culminate in a definitive flight experiment); and (3) to experimentally investigate the vibration sensitivity of liquid bridges under terrestrial and low gravity conditions.
msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

PubMed

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).
Liquid crystal dynamic flow control by bidirectional alignment surface

NASA Astrophysics Data System (ADS)

Li, Y. W.; Lee, C. Y.; Kwok, H. S.

2009-02-01

We investigate the behavior of liquid crystal dynamic flow in a cell with a bidirectional alignment (BDA) surface. Numerical simulations show that with a BDA surface having a pitch comparable to the cell gap d, the liquid crystal dynamic flow direction can be controlled by the driving voltage. Such an effect can be applied to bistable twisted nematic displays without the need for anchoring breaking.
Enhancing the Motor Skills of Children with Autism Spectrum Disorders: A Pool-Based Approach

ERIC Educational Resources Information Center

Lee, Jihyun; Porretta, David L.

2013-01-01

Children with autism spectrum disorders (ASDs) often experience difficulties with motor skill learning and performance. The pool is a unique learning environment that can help children with ASDs learn or improve aquatic skills, fitness, and social skills. A pool-based approach is also aligned with the elements of dynamic systems theory, which…
Fully Anisotropic Rotational Diffusion Tensor from Molecular Dynamics Simulations.

PubMed

Linke, Max; Köfinger, Jürgen; Hummer, Gerhard

2018-05-31

We present a method to calculate the fully anisotropic rotational diffusion tensor from molecular dynamics simulations. Our approach is based on fitting the time-dependent covariance matrix of the quaternions that describe the rigid-body rotational dynamics. Explicit analytical expressions have been derived for the covariances by Favro, which are valid irrespective of the degree of anisotropy. We use these expressions to determine an optimal rotational diffusion tensor from trajectory data. The molecular structures are aligned against a reference by optimal rigid-body superposition. The quaternion covariances can then be obtained directly from the rotation matrices used in the alignment. The rotational diffusion tensor is determined by a fit to the time-dependent quaternion covariances, or directly by Laplace transformation and matrix diagonalization. To quantify uncertainties in the fit, we derive analytical expressions and compare them with the results of Brownian dynamics simulations of anisotropic rotational diffusion. We apply the method to microsecond long trajectories of the Dickerson-Drew B-DNA dodecamer and of horse heart myoglobin. The anisotropic rotational diffusion tensors calculated from simulations agree well with predictions from hydrodynamics.
In-Flight Alignment Using H ∞ Filter for Strapdown INS on Aircraft

PubMed Central

Pei, Fu-Jun; Liu, Xuan; Zhu, Li

2014-01-01

In-flight alignment is an effective way to improve the accuracy and speed of initial alignment for strapdown inertial navigation system (INS). During the aircraft flight, strapdown INS alignment was disturbed by lineal and angular movements of the aircraft. To deal with the disturbances in dynamic initial alignment, a novel alignment method for SINS is investigated in this paper. In this method, an initial alignment error model of SINS in the inertial frame is established. The observability of the system is discussed by piece-wise constant system (PWCS) theory and observable degree is computed by the singular value decomposition (SVD) theory. It is demonstrated that the system is completely observable, and all the system state parameters can be estimated by optimal filter. Then a H ∞ filter was designed to resolve the uncertainty of measurement noise. The simulation results demonstrate that the proposed algorithm can reach a better accuracy under the dynamic disturbance condition. PMID:24511300
Living nanofiber yarn-based woven biotextiles for tendon tissue engineering using cell tri-culture and mechanical stimulation.

PubMed

Wu, Shaohua; Wang, Ying; Streubel, Philipp N; Duan, Bin

2017-10-15

Non-woven nanofibrous scaffolds have been developed for tendon graft application by using electrospinning strategies. However, electrospun nanofibrous scaffolds face some obstacles and limitations, including suboptimal scaffold structure, weak tensile and suture-retention strengths, and compact structure for cell infiltration. In this work, a novel nanofibrous, woven biotextile, fabricated based on electrospun nanofiber yarns, was implemented as a tissue engineered tendon scaffold. Based on our modified electrospinning setup, polycaprolactone (PCL) nanofiber yarns were fabricated with reproducible quality, and were further processed into plain-weaving fabrics interlaced with polylactic acid (PLA) multifilaments. Nonwoven nanofibrous PCL meshes with random or aligned fiber structures were generated using typical electrospinning as comparative counterparts. The woven fabrics contained 3D aligned microstructures with significantly larger pore size and obviously enhanced tensile mechanical properties than their nonwoven counterparts. The biological results revealed that cell proliferation and infiltration, along with the expression of tendon-specific genes by human adipose derived mesenchymal stem cells (HADMSC) and human tenocytes (HT), were significantly enhanced on the woven fabrics compared with those on randomly-oriented or aligned nanofiber meshes. Co-cultures of HADMSC with HT or human umbilical vein endothelial cells (HUVEC) on woven fabrics significantly upregulated the functional expression of most tenogenic markers. HADMSC/HT/HUVEC tri-culture on woven fabrics showed the highest upregulation of most tendon-associated markers than all the other mono- and co-culture groups. Furthermore, we conditioned the tri-cultured constructs with dynamic conditioning and demonstrated that dynamic stretch promoted total collagen secretion and tenogenic differentiation. Our nanofiber yarn-based biotextiles have significant potential to be used as engineered scaffolds to synergize the multiple cell interaction and mechanical stimulation for promoting tendon regeneration. Tendon grafts are essential for the treatment of various tendon-related conditions due to the inherently poor healing capacity of native tendon tissues. In this study, we combined electrospun nanofiber yarns with textile manufacturing strategies to fabricate nanofibrous woven biotextiles with hierarchical features, aligned fibrous topography, and sufficient mechanical properties as tendon tissue engineered scaffolds. Comparing to traditional electrospun random or aligned meshes, our novel nanofibrous woven fabrics possess strong tensile and suture-retention strengths and larger pore size. We also demonstrated that the incorporation of tendon cells and vascular cells promoted the tenogenic differentiation of the engineered tendon constructs, especially under dynamic stretch. This study not only presents a novel tissue engineered tendon scaffold fabrication technique but also provides a useful strategy to promote tendon differentiation and regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
TLM-PSD model for optimization of energy and power density of vertically aligned carbon nanotube supercapacitor

PubMed Central

Ghosh, Arunabha; Le, Viet Thong; Bae, Jung Jun; Lee, Young Hee

2013-01-01

Electrochemical capacitors with fast charging-discharging rates are very promising for hybrid electric vehicle industries including portable electronics. Complicated pore structures have been implemented in active materials to increase energy storage capacity, which often leads to degrade dynamic response of ions. In order to understand this trade-off phenomenon, we report a theoretical model based on transmission line model which is further combined with pore size distribution function. The model successfully explained how pores length, and pore radius of active materials and electrolyte conductivity can affect capacitance and dynamic performance of different capacitors. The powerfulness of the model was confirmed by comparing with experimental results of a micro-supercapacitor consisted of vertically aligned multiwalled carbon nanotubes (v-MWCNTs), which revealed a linear current increase up to 600 Vs−1 scan rate demonstrating an ultrafast dynamic behavior, superior to randomly entangled singlewalled carbon nanotube device, which is clearly explained by the theoretical model. PMID:24145831
Statistical properties of fluctuating enzymes with dynamic cooperativity using a first passage time distribution formalism.

PubMed

Singh, Divya; Chaudhury, Srabanti

2017-04-14

We study the temporal fluctuations in catalytic rates for single enzyme reactions undergoing slow transitions between two active states. We use a first passage time distribution formalism to obtain the closed-form analytical expressions of the mean reaction time and the randomness parameter for reaction schemes where conformational fluctuations are present between two free enzyme conformers. Our studies confirm that the sole presence of free enzyme fluctuations yields a non Michaelis-Menten equation and can lead to dynamic cooperativity. The randomness parameter, which is a measure of the dynamic disorder in the system, converges to unity at a high substrate concentration. If slow fluctuations are present between the enzyme-substrate conformers (off-pathway mechanism), dynamic disorder is present at a high substrate concentration. Our results confirm that the dynamic disorder at a high substrate concentration is determined only by the slow fluctuations between the enzyme-substrate conformers and the randomness parameter is greater than unity. Slow conformational fluctuations between free enzymes are responsible for the emergence of dynamic cooperativity in single enzymes. Our theoretical findings are well supported by comparison with experimental data on the single enzyme beta-galactosidase.
Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

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

Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarilymore » at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.« less
Virus scaffolds as enzyme nano-carriers.

PubMed

Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

2012-07-01

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.
The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

PubMed Central

Francis, Kevin; Sapienza, Paul J.; Lee, Andrew L.; Kohen, Amnon

2016-01-01

Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general. PMID:26813442
Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes.

PubMed

Hay, Sam; Johannissen, Linus O; Hothi, Parvinder; Sutcliffe, Michael J; Scrutton, Nigel S

2012-06-13

The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.

Role of dynamics in enzyme catalysis: substantial versus semantic controversies.

PubMed

Kohen, Amnon

2015-02-17

CONSPECTUS: The role of the enzyme's dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term "protein dynamics" is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is "stored" in a specific protein mode and "used" for catalysis before it can dissipate to its environment (i.e., "nonstatistical dynamics"). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual "induced-fit" shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction's transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions.
Robust temporal alignment of multimodal cardiac sequences

NASA Astrophysics Data System (ADS)

Perissinotto, Andrea; Queirós, Sandro; Morais, Pedro; Baptista, Maria J.; Monaghan, Mark; Rodrigues, Nuno F.; D'hooge, Jan; Vilaça, João. L.; Barbosa, Daniel

2015-03-01

Given the dynamic nature of cardiac function, correct temporal alignment of pre-operative models and intraoperative images is crucial for augmented reality in cardiac image-guided interventions. As such, the current study focuses on the development of an image-based strategy for temporal alignment of multimodal cardiac imaging sequences, such as cine Magnetic Resonance Imaging (MRI) or 3D Ultrasound (US). First, we derive a robust, modality-independent signal from the image sequences, estimated by computing the normalized cross-correlation between each frame in the temporal sequence and the end-diastolic frame. This signal is a resembler for the left-ventricle (LV) volume curve over time, whose variation indicates different temporal landmarks of the cardiac cycle. We then perform the temporal alignment of these surrogate signals derived from MRI and US sequences of the same patient through Dynamic Time Warping (DTW), allowing to synchronize both sequences. The proposed framework was evaluated in 98 patients, which have undergone both 3D+t MRI and US scans. The end-systolic frame could be accurately estimated as the minimum of the image-derived surrogate signal, presenting a relative error of 1.6 +/- 1.9% and 4.0 +/- 4.2% for the MRI and US sequences, respectively, thus supporting its association with key temporal instants of the cardiac cycle. The use of DTW reduces the desynchronization of the cardiac events in MRI and US sequences, allowing to temporally align multimodal cardiac imaging sequences. Overall, a generic, fast and accurate method for temporal synchronization of MRI and US sequences of the same patient was introduced. This approach could be straightforwardly used for the correct temporal alignment of pre-operative MRI information and intra-operative US images.
First-principles approach to calculating energy level alignment at aqueous semiconductor interfaces

DOE PAGES

Kharche, Neerav; Muckerman, James T.; Hybertsen, Mark S.

2014-10-21

A first-principles approach is demonstrated for calculating the relationship between an aqueous semiconductor interface structure and energy level alignment. The physical interface structure is sampled using density functional theory based molecular dynamics, yielding the interface electrostatic dipole. The GW approach from many-body perturbation theory is used to place the electronic band edge energies of the semiconductor relative to the occupied 1 b₁ energy level in water. The application to the specific cases of nonpolar (101¯0 ) facets of GaN and ZnO reveals a significant role for the structural motifs at the interface, including the degree of interface water dissociation andmore » the dynamical fluctuations in the interface Zn-O and O-H bond orientations. As a result, these effects contribute up to 0.5 eV.« less
Heuristic reusable dynamic programming: efficient updates of local sequence alignment.

PubMed

Hong, Changjin; Tewfik, Ahmed H

2009-01-01

Recomputation of the previously evaluated similarity results between biological sequences becomes inevitable when researchers realize errors in their sequenced data or when the researchers have to compare nearly similar sequences, e.g., in a family of proteins. We present an efficient scheme for updating local sequence alignments with an affine gap model. In principle, using the previous matching result between two amino acid sequences, we perform a forward-backward alignment to generate heuristic searching bands which are bounded by a set of suboptimal paths. Given a correctly updated sequence, we initially predict a new score of the alignment path for each contour to select the best candidates among them. Then, we run the Smith-Waterman algorithm in this confined space. Furthermore, our heuristic alignment for an updated sequence shows that it can be further accelerated by using reusable dynamic programming (rDP), our prior work. In this study, we successfully validate "relative node tolerance bound" (RNTB) in the pruned searching space. Furthermore, we improve the computational performance by quantifying the successful RNTB tolerance probability and switch to rDP on perturbation-resilient columns only. In our searching space derived by a threshold value of 90 percent of the optimal alignment score, we find that 98.3 percent of contours contain correctly updated paths. We also find that our method consumes only 25.36 percent of the runtime cost of sparse dynamic programming (sDP) method, and to only 2.55 percent of that of a normal dynamic programming with the Smith-Waterman algorithm.
Role of conformational dynamics in the evolution of novel enzyme function.

PubMed

Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

2018-05-21

The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.
Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens

PubMed Central

Naz, Sadia; Ngo, Tony; Farooq, Umar

2017-01-01

Background The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis. The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Methods Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli, two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. Results High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis. Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket alignments and distance-based maps provide guidelines for drug discovery and repurposing. In addition they also provide recommendations for the relevant model bacteria that may be used for initial drug testing. Discussion Comparing ligand binding sites through sequence identity calculation could be an effective approach to identify conserved orthologs as drug binding pockets have shown higher level of conservation among various species. By using this approach we could avoid the problems associated with full sequence comparison. We identified essential metabolic enzymes among ESKAPE pathogens that share high sequence identity in their putative drug binding pockets (up to 100%), of which known inhibitors can potentially antagonize these identical pockets in the various species in a similar manner. PMID:28948099
Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens.

PubMed

Naz, Sadia; Ngo, Tony; Farooq, Umar; Abagyan, Ruben

2017-01-01

The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis . The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli , two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis . Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket alignments and distance-based maps provide guidelines for drug discovery and repurposing. In addition they also provide recommendations for the relevant model bacteria that may be used for initial drug testing. Comparing ligand binding sites through sequence identity calculation could be an effective approach to identify conserved orthologs as drug binding pockets have shown higher level of conservation among various species. By using this approach we could avoid the problems associated with full sequence comparison. We identified essential metabolic enzymes among ESKAPE pathogens that share high sequence identity in their putative drug binding pockets (up to 100%), of which known inhibitors can potentially antagonize these identical pockets in the various species in a similar manner.
Mechanisms of the Anterior Cruciate Ligament Injury in Sports Activities: A Twenty-Year Clinical Research of 1,700 Athletes

PubMed Central

Kobayashi, Hirokazu; Kanamura, Tomonao; Koshida, Sentaro; Miyashita, Koji; Okado, Tsuruo; Shimizu, Takuya; Yokoe, Kiyoshi

2010-01-01

The mechanisms of anterior cruciate ligament (ACL) injuries are still inconclusive from an epidemiological standpoint. An epidemiological approach in a large sample group over an appropriate period of years will be necessary to enhance the current knowledge of the ACL injury mechanism. The objective of the study was to investigate the ACL injury occurrence in a large sample over twenty years and demonstrate the relationships between the ACL injury occurrence and the dynamic knee alignment at the time of the injury. We investigated the activity, the injury mechanism, and the dynamic knee alignment at the time of the injury in 1,718 patients diagnosed as having the ACL injuries. Regarding the activity at the time of the injury, “competition ”was the most common, accounting for about half of all the injuries. The current result also showed that the noncontact injury was the most common, which was observed especially in many female athletes. Finally, the dynamic alignment of “Knee-in & Toe- out ”(i.e. dynamic knee valgus) was the most common, accounting for about half. These results enhance our understanding of the ACL injury mechanism and may be used to guide future injury prevention strategies. Key points We investigated the situation of ACL injury occurrence, especially dynamic alignments at the time of injury, in 1,718 patients who had visited our institution for surgery and physical therapy for twenty years. Our epidemiological study of the large patient group revealed that “knee-in & toe-out ”alignment was the most frequently seen at the time of the ACL injury. From an epidemiological standpoint, we need to pay much attention to avoiding “Knee-in & Toe-out ”alignment during sports activities. PMID:24149795
Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.
Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

PubMed

Pornbanlualap, Somchai; Chalopagorn, Pornchanok

2011-08-01

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. Copyright © 2011 Elsevier Inc. All rights reserved.
Conformational Plasticity of an Enzyme during Catalysis: Intricate Coupling between Cyclophilin A Dynamics and Substrate Turnover

PubMed Central

McGowan, Lauren C.; Hamelberg, Donald

2013-01-01

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. PMID:23332074
Conformational plasticity of an enzyme during catalysis: intricate coupling between cyclophilin A dynamics and substrate turnover.

PubMed

McGowan, Lauren C; Hamelberg, Donald

2013-01-08

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Molecular dynamics studies of a hexameric purine nucleoside phosphorylase.

PubMed

Zanchi, Fernando Berton; Caceres, Rafael Andrade; Stabeli, Rodrigo Guerino; de Azevedo, Walter Filgueira

2010-03-01

Purine nucleoside phosphorylase (PNP) (EC.2.4.2.1) is an enzyme that catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is involved in purine-salvage pathway and has been proposed as a promising target for design and development of antimalarial and antibacterial drugs. Recent elucidation of the three-dimensional structure of PNP by X-ray protein crystallography left open the possibility of structure-based virtual screening initiatives in combination with molecular dynamics simulations focused on identification of potential new antimalarial drugs. Most of the previously published molecular dynamics simulations of PNP were carried out on human PNP, a trimeric PNP. The present article describes for the first time molecular dynamics simulations of hexameric PNP from Plasmodium falciparum (PfPNP). Two systems were simulated in the present work, PfPNP in ligand free form, and in complex with immucillin and sulfate. Based on the dynamical behavior of both systems the main results related to structural stability and protein-drug interactions are discussed.
Normal Modes Expose Active Sites in Enzymes.

PubMed

Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

2016-12-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.
Normal Modes Expose Active Sites in Enzymes

PubMed Central

Glantz-Gashai, Yitav; Samson, Abraham O.

2016-01-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427
Kinematic Model of Transient Shape-Induced Anisotropy in Dense Granular Flow

NASA Astrophysics Data System (ADS)

Nadler, B.; Guillard, F.; Einav, I.

2018-05-01

Nonspherical particles are ubiquitous in nature and industry, yet previous theoretical models of granular media are mostly limited to systems of spherical particles. The problem is that in systems of nonspherical anisotropic particles, dynamic particle alignment critically affects their mechanical response. To study the tendency of such particles to align, we propose a simple kinematic model that relates the flow to the evolution of particle alignment with respect to each other. The validity of the proposed model is supported by comparison with particle-based simulations for various particle shapes ranging from elongated rice-like (prolate) to flattened lentil-like (oblate) particles. The model shows good agreement with the simulations for both steady-state and transient responses, and advances the development of comprehensive constitutive models for shape-anisotropic particles.
Mechanism of cell alignment in groups of Myxococcus xanthus bacteria

NASA Astrophysics Data System (ADS)

Balgam, Rajesh; Igoshin, Oleg

2015-03-01

Myxococcus xanthus is a model for studying self-organization in bacteria. These flexible cylindrical bacteria move along. In groups, M. xanthus cells align themselves into dynamic cell clusters but the mechanism underlying their formation is unknown. It has been shown that steric interactions can cause alignment in self-propelled hard rods but it is not clear how flexibility and reversals affect the alignment and cluster formation. We have investigated cell alignment process using our biophysical model of M. xanthus cell in an agent-based simulation framework under realistic cell flexibility values. We observed that flexible model cells can form aligned cell clusters when reversals are suppressed but these clusters disappeared when reversals frequency becomes similar to the observed value. However, M. xanthus cells follow slime (polysaccharide gel like material) trails left by other cells and we show that implementing this into our model rescues cell clustering for reversing cells. Our results show that slime following along with periodic cell reversals act as positive feedback to reinforce existing slime trails and recruit more cells. Furthermore, we have observed that mechanical cell alignment combined with slime following is sufficient to explain the distinct clustering patterns of reversing and non-reversing cells as observed in recent experiments. This work is supported by NSF MCB 0845919 and 1411780.
Optofluidic laser for dual-mode sensitive biomolecular detection with a large dynamic range

NASA Astrophysics Data System (ADS)

Wu, Xiang; Oo, Maung Kyaw Khaing; Reddy, Karthik; Chen, Qiushu; Sun, Yuze; Fan, Xudong

2014-04-01

Enzyme-linked immunosorbent assay (ELISA) is a powerful method for biomolecular analysis. The traditional ELISA employing light intensity as the sensing signal often encounters large background arising from non-specific bindings, material autofluorescence and leakage of excitation light, which deteriorates its detection limit and dynamic range. Here we develop the optofluidic laser-based ELISA, where ELISA occurs inside a laser cavity. The laser onset time is used as the sensing signal, which is inversely proportional to the enzyme concentration and hence the analyte concentration inside the cavity. We first elucidate the principle of the optofluidic laser-based ELISA, and then characterize the optofluidic laser performance. Finally, we present the dual-mode detection of interleukin-6 using commercial ELISA kits, where the sensing signals are simultaneously obtained by the traditional and the optofluidic laser-based ELISA, showing a detection limit of 1 fg ml-1 (38 aM) and a dynamic range of 6 orders of magnitude.
Investigating Conversational Dynamics: Interactive Alignment, Interpersonal Synergy, and Collective Task Performance

ERIC Educational Resources Information Center

Fusaroli, Riccardo; Tylén, Kristian

2016-01-01

This study investigates interpersonal processes underlying dialog by comparing two approaches, "interactive alignment" and "interpersonal synergy", and assesses how they predict collective performance in a joint task. While the interactive alignment approach highlights imitative patterns between interlocutors, the synergy…
Inhibitor design strategy based on an enzyme structural flexibility: a case of bacterial MurD ligase.

PubMed

Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-05-27

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

First Molecular Characterization of Hypoderma actaeon in Cattle and Red Deer (Cervus elaphus) in Portugal.

PubMed

Ahmed, Haroon; Sousa, Sérgio Ramalho; Simsek, Sami; Anastácio, Sofia; Kilinc, Seyma Gunyakti

2017-12-01

Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.
Positioning (Mis)Aligned: The (Un)Making of Intercultural Asynchronous Computer-Mediated Communication

ERIC Educational Resources Information Center

Wu, Zhiwei

2018-01-01

Framed from positioning theory and dynamic systems theory, the paper reports on a naturalistic study involving four Chinese participants and their American peers in an intercultural asynchronous computer-mediated communication (ACMC) activity. Based on the moment-by-moment analysis and triangulation of forum posts, reflective essays, and…
Automatic Construction of English/Chinese Parallel Corpora.

ERIC Educational Resources Information Center

Yang, Christopher C.; Li, Kar Wing

2003-01-01

Discussion of multilingual corpora and cross-lingual information retrieval focuses on research that constructed English/Chinese parallel corpus automatically from the World Wide Web. Presents an alignment method which is based on dynamic programming to identify one-to-one Chinese and English title pairs and discusses results of experiments…
LIMAO: Cross-platform software for simulating laser-induced alignment and orientation dynamics of linear-, symmetric- and asymmetric tops

NASA Astrophysics Data System (ADS)

Szidarovszky, Tamás; Jono, Maho; Yamanouchi, Kaoru

2018-07-01

A user-friendly and cross-platform software called Laser-Induced Molecular Alignment and Orientation simulator (LIMAO) has been developed. The program can be used to simulate within the rigid rotor approximation the rotational dynamics of gas phase molecules induced by linearly polarized intense laser fields at a given temperature. The software is implemented in the Java and Mathematica programming languages. The primary aim of LIMAO is to aid experimental scientists in predicting and analyzing experimental data representing laser-induced spatial alignment and orientation of molecules.
Dynamic programming algorithms for biological sequence comparison.

PubMed

Pearson, W R; Miller, W

1992-01-01

Efficient dynamic programming algorithms are available for a broad class of protein and DNA sequence comparison problems. These algorithms require computer time proportional to the product of the lengths of the two sequences being compared [O(N2)] but require memory space proportional only to the sum of these lengths [O(N)]. Although the requirement for O(N2) time limits use of the algorithms to the largest computers when searching protein and DNA sequence databases, many other applications of these algorithms, such as calculation of distances for evolutionary trees and comparison of a new sequence to a library of sequence profiles, are well within the capabilities of desktop computers. In particular, the results of library searches with rapid searching programs, such as FASTA or BLAST, should be confirmed by performing a rigorous optimal alignment. Whereas rapid methods do not overlook significant sequence similarities, FASTA limits the number of gaps that can be inserted into an alignment, so that a rigorous alignment may extend the alignment substantially in some cases. BLAST does not allow gaps in the local regions that it reports; a calculation that allows gaps is very likely to extend the alignment substantially. Although a Monte Carlo evaluation of the statistical significance of a similarity score with a rigorous algorithm is much slower than the heuristic approach used by the RDF2 program, the dynamic programming approach should take less than 1 hr on a 386-based PC or desktop Unix workstation. For descriptive purposes, we have limited our discussion to methods for calculating similarity scores and distances that use gap penalties of the form g = rk. Nevertheless, programs for the more general case (g = q+rk) are readily available. Versions of these programs that run either on Unix workstations, IBM-PC class computers, or the Macintosh can be obtained from either of the authors.
Multiscale simulations give insight into the hydrogen in and out pathways of [NiFe]-hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans.

PubMed

Oteri, Francesco; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

2014-12-04

[NiFe]-hydrogenases catalyze the cleavage of molecular hydrogen into protons and electrons and represent promising tools for H2-based technologies such as biofuel cells. However, many aspects of these enzymes remain to be understood, in particular how the catalytic center can be protected from irreversible inactivation by O2. In this work, we combined homology modeling, all-atom molecular dynamics, and coarse-grain Brownian dynamics simulations to investigate and compare the dynamic and mechanical properties of two [NiFe]-hydrogenases: the soluble O2-sensitive enzyme from Desulfovibrio fructosovorans, and the O2-tolerant membrane-bound hydrogenase from Aquifex aeolicus. We investigated the diffusion pathways of H2 from the enzyme surface to the central [NiFe] active site, and the possible proton pathways that are used to evacuate hydrogen after the oxidation reaction. Our results highlight common features of the two enzymes, such as a Val/Leu/Arg triad of key residues that controls ligand migration and substrate access in the vicinity of the active site, or the key role played by a Glu residue for proton transfer after hydrogen oxidation. We show specificities of each hydrogenase regarding the enzymes internal tunnel network or the proton transport pathways.
Conformational Sub-states and Populations in Enzyme Catalysis

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

Agarwal, Pratul K; Doucet, Nicholas; Chennubholta, C

reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme substrate complex allow reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surroundingmore » environment. Areas of low energy along the landscape form conformational sub-states, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling, and nuclear magnetic resonance have been developed for quantitative characterization of these sub-states and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.« less
Further Validation of Simulated Dynamic Interface Testing Techniques as a Tool in the Forecasting of Air Vehicle Deck Limits

DTIC Science & Technology

2010-01-01

UAV Autonomy program which includes intelligent reasoning for autonomy, technologies to enhance see and avoid capabilities, object identification ...along the ship’s base recovery course (BRC). The pilot then flies toward the stern of the ship, aligning his approach path with the ship’s lineup line...quiescent point identification . CONCLUSIONS The primary goal for conducting dynamic interface analysis is to expand existing operating envelopes and
Multi-exposure high dynamic range image synthesis with camera shake correction

NASA Astrophysics Data System (ADS)

Li, Xudong; Chen, Yongfu; Jiang, Hongzhi; Zhao, Huijie

2017-10-01

Machine vision plays an important part in industrial online inspection. Owing to the nonuniform illuminance conditions and variable working distances, the captured image tends to be over-exposed or under-exposed. As a result, when processing the image such as crack inspection, the algorithm complexity and computing time increase. Multiexposure high dynamic range (HDR) image synthesis is used to improve the quality of the captured image, whose dynamic range is limited. Inevitably, camera shake will result in ghost effect, which blurs the synthesis image to some extent. However, existed exposure fusion algorithms assume that the input images are either perfectly aligned or captured in the same scene. These assumptions limit the application. At present, widely used registration based on Scale Invariant Feature Transform (SIFT) is usually time consuming. In order to rapidly obtain a high quality HDR image without ghost effect, we come up with an efficient Low Dynamic Range (LDR) images capturing approach and propose a registration method based on ORiented Brief (ORB) and histogram equalization which can eliminate the illumination differences between the LDR images. The fusion is performed after alignment. The experiment results demonstrate that the proposed method is robust to illumination changes and local geometric distortion. Comparing with other exposure fusion methods, our method is more efficient and can produce HDR images without ghost effect by registering and fusing four multi-exposure images.
Interactions Between Mineral Surfaces, Substrates, Enzymes, and Microbes Result in Hysteretic Temperature Sensitivities and Microbial Carbon Use Efficiencies and Weaker Predicted Carbon-Climate Feedbacks

NASA Astrophysics Data System (ADS)

Riley, W. J.; Tang, J.

2014-12-01

We hypothesize that the large observed variability in decomposition temperature sensitivity and carbon use efficiency arises from interactions between temperature, microbial biogeochemistry, and mineral surface sorptive reactions. To test this hypothesis, we developed a numerical model that integrates the Dynamic Energy Budget concept for microbial physiology, microbial trait-based community structure and competition, process-specific thermodynamically based temperature sensitivity, a non-linear mineral sorption isotherm, and enzyme dynamics. We show, because mineral surfaces interact with substrates, enzymes, and microbes, both temperature sensitivity and microbial carbon use efficiency are hysteretic and highly variable. Further, by mimicking the traditional approach to interpreting soil incubation observations, we demonstrate that the conventional labile and recalcitrant substrate characterization for temperature sensitivity is flawed. In a 4 K temperature perturbation experiment, our fully dynamic model predicted more variable but weaker carbon-climate feedbacks than did the static temperature sensitivity and carbon use efficiency model when forced with yearly, daily, and hourly variable temperatures. These results imply that current earth system models likely over-estimate the response of soil carbon stocks to global warming.
Dynamic protein assembly by programmable DNA strand displacement.

PubMed

Chen, Rebecca P; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-04-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.
Dynamic protein assembly by programmable DNA strand displacement

NASA Astrophysics Data System (ADS)

Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-03-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.
Enzyme-free nucleic acid dynamical systems.

PubMed

Srinivas, Niranjan; Parkin, James; Seelig, Georg; Winfree, Erik; Soloveichik, David

2017-12-15

Chemistries exhibiting complex dynamics-from inorganic oscillators to gene regulatory networks-have been long known but either cannot be reprogrammed at will or rely on the sophisticated enzyme chemistry underlying the central dogma. Can simpler molecular mechanisms, designed from scratch, exhibit the same range of behaviors? Abstract chemical reaction networks have been proposed as a programming language for complex dynamics, along with their systematic implementation using short synthetic DNA molecules. We developed this technology for dynamical systems by identifying critical design principles and codifying them into a compiler automating the design process. Using this approach, we built an oscillator containing only DNA components, establishing that Watson-Crick base-pairing interactions alone suffice for complex chemical dynamics and that autonomous molecular systems can be designed via molecular programming languages. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Adaptable liquid crystal elastomers with transesterification-based bond exchange reactions.

PubMed

Hanzon, Drew W; Traugutt, Nicholas A; McBride, Matthew K; Bowman, Christopher N; Yakacki, Christopher M; Yu, Kai

2018-02-14

Adaptable liquid crystal elastomers (LCEs) have recently emerged to provide a new and robust method to program monodomain LCE samples. When a constant stress is applied with active bond exchange reactions (BERs), polymer chains and mesogens gradually align in the strain direction. Mesogen alignment is maintained after removing the BER stimulus (e.g. by lowering the temperature) and the programmed LCE samples exhibit free-standing two-way shape switching behavior. Here, a new adaptable main-chain LCE system was developed with thermally induced transesterification BERs. The network combines the conventional properties of LCEs, such as an isotropic phase transition and soft elasticity, with the dynamic features of adaptable network polymers, which are malleable to stress relaxation due to the BERs. Polarized Fourier transform infrared measurements confirmed the alignment of polymer chains and mesogens after strain-induced programming. The influence of the creep stress, temperature, and time on the strain amplitude of two-way shape switching was examined. The LCE network demonstrates an innovative feature of reprogrammability, where the reversible shape-switching memory of programmed LCEs is readily deleted by free-standing heating as random BERs disrupt the mesogen alignment, so LCEs are reprogrammed after returning to the polydomain state. Due to the dynamic nature of the LCE network, it also exhibits a surface welding effect and can be fully dissolved in the organic solvent, which might be utilized for green and sustainable recycling of LCEs.
Liquid crystal-based biosensor with backscattering interferometry: A quantitative approach.

PubMed

Khan, Mashooq; Park, Soo-Young

2017-01-15

We developed a new technology that uses backscattering interferometry (BSI) to quantitatively measure nematic liquid crystal (NLC)-based biosensors, those usually relied on texture reading for on/off signals. The LC-based BSI comprised an octadecyltrichlorosilane (OTS)-coated square capillary filled with 4-cyano-4'-pentylbiphenyl (5CB, a nematic LC at room temperature). The LC/water interface in the capillary was functionalized by a coating of poly(acrylicacid-b-4-cyanobiphenyl-4'-oxyundecylacrylate) (PAA-b-LCP) and immobilized with the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) through covalent linkage to the PAA chains (5CB PAA-GOx:HRP ) for glucose detection. Laser irradiation of the LC near the LC/water interface resulted in backscattered fringes with high contrast. The change in the spatial position of the fringes (because of the change in the orientation of the LC caused by the GOx:HRP enzymatic reaction of glucose) altered the output voltage of the photodetector when its active area was aligned with the edge of one of the fringes. The change in the intensity at the photodetector allowed the detection limit of the instrument to be as low as 0.008mM with a linear range of 0.02-9mM in a short response time (~60s). This LC-based BSI technique allows for quantitative, sensitive, selective, reproducible, easily obtainable, and interference-free detection in a large linear dynamic range and for practical applications with human serum. Copyright Â© 2016 Elsevier B.V. All rights reserved.
Representing and comparing protein structures as paths in three-dimensional space

PubMed Central

Zhi, Degui; Krishna, S Sri; Cao, Haibo; Pevzner, Pavel; Godzik, Adam

2006-01-01

Background Most existing formulations of protein structure comparison are based on detailed atomic level descriptions of protein structures and bypass potential insights that arise from a higher-level abstraction. Results We propose a structure comparison approach based on a simplified representation of proteins that describes its three-dimensional path by local curvature along the generalized backbone of the polypeptide. We have implemented a dynamic programming procedure that aligns curvatures of proteins by optimizing a defined sum turning angle deviation measure. Conclusion Although our procedure does not directly optimize global structural similarity as measured by RMSD, our benchmarking results indicate that it can surprisingly well recover the structural similarity defined by structure classification databases and traditional structure alignment programs. In addition, our program can recognize similarities between structures with extensive conformation changes that are beyond the ability of traditional structure alignment programs. We demonstrate the applications of procedure to several contexts of structure comparison. An implementation of our procedure, CURVE, is available as a public webserver. PMID:17052359
Dynamic mechanical analysis and high strain-rate energy absorption characteristics of vertically aligned carbon nanotube reinforced woven fiber-glass composites

USDA-ARS?s Scientific Manuscript database

The dynamic mechanical behavior and energy absorption characteristics of nano-enhanced functionally graded composites, consisting of 3 layers of vertically aligned carbon nanotube (VACNT) forests grown on woven fiber-glass (FG) layer and embedded within 10 layers of woven FG, with polyester (PE) and...
Protein dynamics and enzyme catalysis: insights from simulations.

PubMed

McGeagh, John D; Ranaghan, Kara E; Mulholland, Adrian J

2011-08-01

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. Copyright © 2010 Elsevier B.V. All rights reserved.
Disruption of sheet-like structures in Alfvénic turbulence by magnetic reconnection

NASA Astrophysics Data System (ADS)

Mallet, A.; Schekochihin, A. A.; Chandran, B. D. G.

2017-07-01

We propose a mechanism whereby the intense, sheet-like structures naturally formed by dynamically aligning Alfvénic turbulence are destroyed by magnetic reconnection at a scale \\hat{λ }_D, larger than the dissipation scale predicted by models of intermittent, dynamically aligning turbulence. The reconnection process proceeds in several stages: first, a linear tearing mode with N magnetic islands grows and saturates, and then the X-points between these islands collapse into secondary current sheets, which then reconnect until the original structure is destroyed. This effectively imposes an upper limit on the anisotropy of the structures within the perpendicular plane, which means that at scale \\hat{λ }_D the turbulent dynamics change: at scales larger than \\hat{λ }_D, the turbulence exhibits scale-dependent dynamic alignment and a spectral index approximately equal to -3/2, while at scales smaller than \\hat{λ }_D, the turbulent structures undergo a succession of disruptions due to reconnection, limiting dynamic alignment, steepening the effective spectral index and changing the final dissipation scale. The scaling of \\hat{λ }_D with the Lundquist (magnetic Reynolds) number S_{L_\\perp } depends on the order of the statistics being considered, and on the specific model of intermittency; the transition between the two regimes in the energy spectrum is predicted at approximately \\hat{λ }_D˜ S_{L_\\perp }^{-0.6}. The spectral index below \\hat{λ }_D is bounded between -5/3 and -2.3. The final dissipation scale is at \\hat{λ }_{η ,∞}˜ S_{L_\\perp }^{-3/4}, the same as the Kolmogorov scale arising in theories of turbulence that do not involve scale-dependent dynamic alignment.
The dynamic basis of energy transduction in enzymes.

PubMed

Somogyi, B; Welch, G R; Damjanovich, S

1984-09-06

The most important idea underlying our treatment herein is the unity of the enzyme molecule and the medium. Appreciation of this relationship is vital, if enzymology is to graduate from its present reductionistic status to a more holistic posture. Enzymes are biological entities firstly, and isolated objects of physicochemical analysis secondly. Perhaps the most crucial 'biological lesson', particularly apropos of enzymes in intermediary metabolism, concerns the 'cytosociology' of enzyme action in vivo [94,128]. The natural habitat of many enzymes in the living cell is far different from that in bulk aqueous solution in vitro. In order to obtain a real grasp of the nature of enzyme function, one must ultimately couch enzymology in concepts emerging from contemporary cell biology [95]. Notwithstanding, analysis precedes synthesis; and one must needs begin with the individual enzyme molecule. The trenchant efforts of the physical chemist and the organic chemist have produced a wealth of information on the nature of the binding and catalytic events at the enzyme active site. While it is not yet possible to explain precisely the complete sequence of events in the catalytic process, nevertheless, the basic mechanisms by which enzymes effect catalysis (i.e., reduce activation energy) now seem apparent [81,129]. The new frontier is to be found, in exploring the dynamic role of the protein matrix [17]. Not only does the protein provide the 3-D scaffolding for active-site processes, but, more importantly, it serves as the local solvent for the bound chemical subsystem. Thus, the dynamical aspects of enzyme catalysis (for thermally based systems) must arise from the fluctuational properties of the protein molecule. This notion is the common denominator in all of the models in subsection IIC. It is the anisotropic nature of this fluctuational behavior, which would characterize the energy-transduction phenomenon leading to localized catalytic events at the active-site. In Section III we attempted to show that all of the various enzyme models contribute pieces to a single, all-embracing jig-saw puzzle. Some models focus on the dynamical properties of the protein per se, whereas others deal with the stochastic aspects of protein-solvent interaction. The two approaches are complementary, as are mutually interlocking pieces of a puzzle. The ultimate picture depicted by this 'jig-saw puzzle' is still somewhat vague--owing to the present paucity of empirical information on protein motions.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification, expression and characterization of an R-ω-transaminase from Capronia semiimmersa.

PubMed

Iglesias, César; Panizza, Paola; Rodriguez Giordano, Sonia

2017-07-01

Chiral amines are essential precursors in the production of biologically active compounds, including several important drugs. Among the biocatalytic strategies that have been developed for their synthesis, the use of ω-transaminases (ω-TA) appears as an attractive alternative allowing the stereoselective amination of prochiral ketones. However, the problems associated with narrow substrate specificity, unfavourable reaction equilibrium and expensive amine donors still hamper its industrial application. The search for novel enzymes from nature can contribute to expand the catalytic repertoire of ω-TA and help to circumvent some of these problems. A genome mining approach, based on the work described by Höhne et al., was applied for selection of potential R-ω-TA. Additional criteria were used to select an enzyme that differs from previously described ones. A candidate R-ω-TA from Capronia semiimmersa was selected, cloned and expressed in Escherichia coli. Interestingly, alignment of this enzyme with previously reported TA sequences revealed the presence of two additional amino acid residues in a loop close to the active site. The impact of this change was analysed with a structural model based on crystallized R-ω-TAs. Analysis of the substrate specificity of R-ω-TA from C. semiimmersa indicates that it accepts a diversity of ketones as substrates yielding the corresponding amine with good yields and excellent enantioselectivity. The expressed enzyme accepts isopropylamine as amine donor what makes it suitable for industrial processes.
Elastic Characterization of Transversely Isotropic Soft Materials by Dynamic Shear and Asymmetric Indentation

PubMed Central

Namani, R.; Feng, Y.; Okamoto, R. J.; Jesuraj, N.; Sakiyama-Elbert, S. E.; Genin, G. M.; Bayly, P. V.

2012-01-01

The mechanical characterization of soft anisotropic materials is a fundamental challenge because of difficulties in applying mechanical loads to soft matter and the need to combine information from multiple tests. A method to characterize the linear elastic properties of transversely isotropic soft materials is proposed, based on the combination of dynamic shear testing (DST) and asymmetric indentation. The procedure was demonstrated by characterizing a nearly incompressible transversely isotropic soft material. A soft gel with controlled anisotropy was obtained by polymerizing a mixture of fibrinogen and thrombin solutions in a high field magnet (B = 11.7 T); fibrils in the resulting gel were predominantly aligned parallel to the magnetic field. Aligned fibrin gels were subject to dynamic (20–40 Hz) shear deformation in two orthogonal directions. The shear storage modulus was 1.08 ± 0. 42 kPa (mean ± std. dev.) for shear in a plane parallel to the dominant fiber direction, and 0.58 ± 0.21 kPa for shear in the plane of isotropy. Gels were indented by a rectangular tip of a large aspect ratio, aligned either parallel or perpendicular to the normal to the plane of transverse isotropy. Aligned fibrin gels appeared stiffer when indented with the long axis of a rectangular tip perpendicular to the dominant fiber direction. Three-dimensional numerical simulations of asymmetric indentation were used to determine the relationship between direction-dependent differences in indentation stiffness and material parameters. This approach enables the estimation of a complete set of parameters for an incompressible, transversely isotropic, linear elastic material. PMID:22757501
Study on Dynamic Alignment Technology of COIL Resonator

NASA Astrophysics Data System (ADS)

Xiong, M. D.; Zou, X. J.; Guo, J. H.; Jia, S. N.; Zhang2, Z. B.

2006-10-01

The performance of great power chemical oxygen-iodine laser (COIL) beam is decided mostly by resonator mirror maladjustment and environment vibration. To improve the performance of light beam, an auto-alignment device is used in COIL resonator, the device can keep COIL resonator collimating by adjusting the optical components of resonator. So the coupling model of COIL resonator is present. The multivariable self study fuzzy uncoupling arithmetic and six-dimensional micro drive technology are used to design a six-input-three-output uncoupling controller, resulting in the realization of the high precision dynamic alignment. The experiments indicate that the collimating range of this system is 8 mrad, precision is 5 urad and frequency response is 20Hz, which meet the demand of resonator alignment system.
Some Alignment Considerations for the Next Linear Collider

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

Ruland, R

Next Linear Collider type accelerators require a new level of alignment quality. The relative alignment of these machines is to be maintained in an error envelope dimensioned in micrometers and for certain parts in nanometers. In the nanometer domain our terra firma cannot be considered monolithic but compares closer to jelly. Since conventional optical alignment methods cannot deal with the dynamics and cannot approach the level of accuracy, special alignment and monitoring techniques must be pursued.
Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

PubMed

Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

2017-09-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.
Dynamic modes of the flipped-out cytosine during HhaI methyltransferase-DNA interactions in solution.

PubMed Central

Klimasauskas, S; Szyperski, T; Serva, S; Wüthrich, K

1998-01-01

Flipping of a nucleotide out of a B-DNA helix into the active site of an enzyme has been observed for the HhaI and HaeIII cytosine-5 methyltransferases (M.HhaI and M.HaeIII) and for numerous DNA repair enzymes. Here we studied the base flipping motions in the binary M. HhaI-DNA and the ternary M.HhaI-DNA-cofactor systems in solution. Two 5-fluorocytosines were introduced into the DNA in the places of the target cytosine and, as an internal control, a cytosine positioned two nucleotides upstream of the recognition sequence 5'-GCGC-3'. The 19F NMR spectra combined with gel mobility data show that interaction with the enzyme induces partition of the target base among three states, i.e. stacked in the B-DNA, an ensemble of flipped-out forms and the flipped-out form locked in the enzyme active site. Addition of the cofactor analogue S-adenosyl-L-homocysteine greatly enhances the trapping of the target cytosine in the catalytic site. Distinct dynamic modes of the target cytosine have thus been identified along the reaction pathway, which includes novel base-flipping intermediates that were not observed in previous X-ray structures. The new data indicate that flipping of the target base out of the DNA helix is not dependent on binding of the cytosine in the catalytic pocket of M.HhaI, and suggest an active role of the enzyme in the opening of the DNA duplex. PMID:9427765
Self-organized sorting limits behavioral variability in swarms

PubMed Central

Copenhagen, Katherine; Quint, David A.; Gopinathan, Ajay

2016-01-01

Swarming is a phenomenon where collective motion arises from simple local interactions between typically identical individuals. Here, we investigate the effects of variability in behavior among the agents in finite swarms with both alignment and cohesive interactions. We show that swarming is abolished above a critical fraction of non-aligners who do not participate in alignment. In certain regimes, however, swarms above the critical threshold can dynamically reorganize and sort out excess non-aligners to maintain the average fraction close to the critical value. This persists even in swarms with a distribution of alignment interactions, suggesting a simple, robust and efficient mechanism that allows heterogeneously mixed populations to naturally regulate their composition and remain in a collective swarming state or even differentiate among behavioral phenotypes. We show that, for evolving swarms, this self-organized sorting behavior can couple to the evolutionary dynamics leading to new evolutionarily stable equilibrium populations set by the physical swarm parameters. PMID:27550316
Self-organized sorting limits behavioral variability in swarms

NASA Astrophysics Data System (ADS)

Copenhagen, Katherine; Quint, David A.; Gopinathan, Ajay

2016-08-01

Swarming is a phenomenon where collective motion arises from simple local interactions between typically identical individuals. Here, we investigate the effects of variability in behavior among the agents in finite swarms with both alignment and cohesive interactions. We show that swarming is abolished above a critical fraction of non-aligners who do not participate in alignment. In certain regimes, however, swarms above the critical threshold can dynamically reorganize and sort out excess non-aligners to maintain the average fraction close to the critical value. This persists even in swarms with a distribution of alignment interactions, suggesting a simple, robust and efficient mechanism that allows heterogeneously mixed populations to naturally regulate their composition and remain in a collective swarming state or even differentiate among behavioral phenotypes. We show that, for evolving swarms, this self-organized sorting behavior can couple to the evolutionary dynamics leading to new evolutionarily stable equilibrium populations set by the physical swarm parameters.
Relationships between field-aligned currents, electric fields, and particle precipitation as observed by Dynamics Explorer-2

NASA Technical Reports Server (NTRS)

Sugiura, M.; Iyemori, T.; Hoffman, R. A.; Maynard, N. C.; Burch, J. L.; Winningham, J. D.

1984-01-01

The relationships between field-aligned currents, electric fields, and particle fluxes are determined using observations from the polar orbiting low-altitude satellite Dynamics Explorer-2. It is shown that the north-south electric field and the east-west magnetic field components are usually highly correlated in the field-aligned current regions. This proportionality observationally proves that the field-aligned current equals the divergence of the height-integrated ionospheric Pedersen current in the meridional plane to a high degree of approximation. As a general rule, in the evening sector the upward field-aligned currents flow in the boundary plasma sheet region and the downward currents flow in the central plasma sheet region. The current densities determined independently from the plasma and magnetic field measurements are compared. Although the current densities deduced from the two methods are in general agreement, the degree and extent of the agreement vary in individual cases.
Relationships between field-aligned currents, electric fields and particle precipitation as observed by dynamics Explorer-2

NASA Technical Reports Server (NTRS)

Sugiura, M.; Iyemori, T.; Hoffman, R. A.; Maynard, N. C.; Burch, J. L.; Winningham, J. D.

1983-01-01

The relationships between field-aligned currents, electric fields, and particle fluxes are determined using observations from the polar orbiting low-altitude satellite Dynamics Explorer-2. It is shown that the north-south electric field and the east-west magnetic field components are usually highly correlated in the field-aligned current regions. This proportionality observationally proves that the field-aligned current equals the divergence of the height-integrated ionospheric Pedersen current in the meridional plane to a high degree of approximation. As a general rule, in the evening sector the upward field-aligned currents flow in the boundary plasma sheet region and the downward currents flow in the central plasma sheet region. The current densities determined independently from the plasma and magnetic field measurements are compared. Although the current densities deduced from the two methods are in general agreement, the degree and extent of the agreement vary in individual cases.
Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

PubMed Central

Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392
Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

PubMed

Ramkissoon, Kevin R; Miller, Jennifer K; Ojha, Sunil; Watson, Douglas S; Bomar, Martha G; Galande, Amit K; Shearer, Alexander G

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.
The impact of static stress change, dynamic stress change, and the background stress on aftershock focal mechanisms

USGS Publications Warehouse

Hardebeck, Jeanne L.

2014-01-01

The focal mechanisms of earthquakes in Southern California before and after four M ≥ 6.7 main shocks provide insight into how fault systems respond to stress and changes in stress. The main shock static stress changes have two observed impacts on the seismicity: changing the focal mechanisms in a given location to favor those aligned with the static stress change and changing the spatial distribution of seismicity to favor locations where the static stress change aligns with the background stress. The aftershock focal mechanisms are significantly aligned with the static stress changes for absolute stress changes of ≥ 0.02 MPa, for up to ~20 years following the main shock. The dynamic stress changes have similar, although smaller, effects on the local focal mechanisms and the spatial seismicity distribution. Dynamic stress effects are best observed at long periods (30–60 s) and for metrics based on repeated stress cycling in the same direction. This implies that dynamic triggering operates, at least in part, through cyclic shear stress loading in the direction of fault slip. The background stress also strongly controls both the preshock and aftershock mechanisms. While most aftershock mechanisms are well oriented in the background stress field, 10% of aftershocks are identified as poorly oriented outliers, which may indicate limited heterogeneity in the postmain shock stress field. The fault plane orientations of the outliers are well oriented in the background stress, while their slip directions are not, implying that the background stress restricts the distribution of available fault planes.
Dynamics and design principles of a basic regulatory architecture controlling metabolic pathways.

PubMed

Chin, Chen-Shan; Chubukov, Victor; Jolly, Emmitt R; DeRisi, Joe; Li, Hao

2008-06-17

The dynamic features of a genetic network's response to environmental fluctuations represent essential functional specifications and thus may constrain the possible choices of network architecture and kinetic parameters. To explore the connection between dynamics and network design, we have analyzed a general regulatory architecture that is commonly found in many metabolic pathways. Such architecture is characterized by a dual control mechanism, with end product feedback inhibition and transcriptional regulation mediated by an intermediate metabolite. As a case study, we measured with high temporal resolution the induction profiles of the enzymes in the leucine biosynthetic pathway in response to leucine depletion, using an automated system for monitoring protein expression levels in single cells. All the genes in the pathway are known to be coregulated by the same transcription factors, but we observed drastically different dynamic responses for enzymes upstream and immediately downstream of the key control point-the intermediate metabolite alpha-isopropylmalate (alphaIPM), which couples metabolic activity to transcriptional regulation. Analysis based on genetic perturbations suggests that the observed dynamics are due to differential regulation by the leucine branch-specific transcription factor Leu3, and that the downstream enzymes are strictly controlled and highly expressed only when alphaIPM is available. These observations allow us to build a simplified mathematical model that accounts for the observed dynamics and can correctly predict the pathway's response to new perturbations. Our model also suggests that transient dynamics and steady state can be separately tuned and that the high induction levels of the downstream enzymes are necessary for fast leucine recovery. It is likely that principles emerging from this work can reveal how gene regulation has evolved to optimize performance in other metabolic pathways with similar architecture.
Resolving the multiple sequence alignment problem using biogeography-based optimization with multiple populations.

PubMed

Zemali, El-Amine; Boukra, Abdelmadjid

2015-08-01

The multiple sequence alignment (MSA) is one of the most challenging problems in bioinformatics, it involves discovering similarity between a set of protein or DNA sequences. This paper introduces a new method for the MSA problem called biogeography-based optimization with multiple populations (BBOMP). It is based on a recent metaheuristic inspired from the mathematics of biogeography named biogeography-based optimization (BBO). To improve the exploration ability of BBO, we have introduced a new concept allowing better exploration of the search space. It consists of manipulating multiple populations having each one its own parameters. These parameters are used to build up progressive alignments allowing more diversity. At each iteration, the best found solution is injected in each population. Moreover, to improve solution quality, six operators are defined. These operators are selected with a dynamic probability which changes according to the operators efficiency. In order to test proposed approach performance, we have considered a set of datasets from Balibase 2.0 and compared it with many recent algorithms such as GAPAM, MSA-GA, QEAMSA and RBT-GA. The results show that the proposed approach achieves better average score than the previously cited methods.
Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production.

PubMed

Chen, Zhen; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-07-20

Deregulation of allosteric inhibition of enzymes is a challenge for strain engineering and has been achieved so far primarily by random mutation and trial-and-error. In this work, we used aspartokinase, an important allosteric enzyme for industrial amino acids production, to demonstrate a predictive approach that combines protein dynamics and evolution for a rational reengineering of enzyme allostery. Molecular dynamic simulation of aspartokinase III (AK3) from Escherichia coli and statistical coupling analysis of protein sequences of the aspartokinase family allowed to identify a cluster of residues which are correlated during protein motion and coupled during the evolution. This cluster of residues forms an interconnected network mediating the allosteric regulation, including most of the previously reported positions mutated in feedback insensitive AK3 mutants. Beyond these mutation positions, we have successfully constructed another twelve targeted mutations of AK3 desensitized toward lysine inhibition. Six threonine-insensitive mutants of aspartokinase I-homoserine dehydrogenase I (AK1-HD1) were also created based on the predictions. The proposed approach can be widely applied for the deregulation of other allosteric enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.
Microfluidic flow rate detection based on integrated optical fiber cantilever.

PubMed

Lien, Victor; Vollmer, Frank

2007-10-01

We demonstrate an integrated microfluidic flow sensor with ultra-wide dynamic range, suitable for high throughput applications such as flow cytometry and particle sorting/counting. A fiber-tip cantilever transduces flow rates to optical signal readout, and we demonstrate a dynamic range from 0 to 1500 microL min(-1) for operation in water. Fiber-optic sensor alignment is guided by preformed microfluidic channels, and the dynamic range can be adjusted in a one-step chemical etch. An overall non-linear response is attributed to the far-field angular distribution of single-mode fiber output.
The Self-Organizing Psyche: Nonlinear and Neurobiological Contributions to Psychoanalysis

NASA Astrophysics Data System (ADS)

Stein, A. H.

Sigmund Freud attempted to align nineteenth century biology (and the dynamically conservative, continuous, Newtonian mechanics that underlie it) with discontinuous conscious experience. His tactics both set the future course for psychoanalytic development and introduced seemingly intractable complications into its metapsychology. In large part, these arose from what we now recognize were biological errors and dynamical oversimplifications amid his physical assumptions. Their correction, brought about by integrating nonlinear dynamics and neuro-biological research findings with W. Bion's reading of metapsychology, fundamentally supports a psychoanalysis based upon D. W. Winnicott's ideas surrounding play within transitional space.
EGenBio: A Data Management System for Evolutionary Genomics and Biodiversity

PubMed Central

Nahum, Laila A; Reynolds, Matthew T; Wang, Zhengyuan O; Faith, Jeremiah J; Jonna, Rahul; Jiang, Zhi J; Meyer, Thomas J; Pollock, David D

2006-01-01

Background Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed Evolutionary Genomics and Biodiversity (EGenBio; ) to begin to address this. Description EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs. Conclusion EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity. PMID:17118150
A Mathematical Optimization Problem in Bioinformatics

ERIC Educational Resources Information Center

Heyer, Laurie J.

2008-01-01

This article describes the sequence alignment problem in bioinformatics. Through examples, we formulate sequence alignment as an optimization problem and show how to compute the optimal alignment with dynamic programming. The examples and sample exercises have been used by the author in a specialized course in bioinformatics, but could be adapted…

Carrier dynamics and recombination mechanisms in staggered-alignment heterostructures

NASA Astrophysics Data System (ADS)

Wilson, Barbara A.

1988-08-01

The experimental and theoretical work on carrier dynamics and recombination mechanisms in semiconductor heterostructures with staggered type II alignments is reviewed. Examples from the literature are discussed for each of the III-V, II-VI, and IV-VI systems, as well as cross-column examples, with a focus on AlGaAs structures. The key optical properties which have benn identified as signatures of staggered-alignment behavior are summarized. A discussion of other epitaxial systems likely to exhibit staggered lineups is presented, and additional experimental and theoretical work is suggested, which could increase understanding of staggered-system behavior.
Dissipative Dynamics of Enzymes

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

2014-11-01

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C ; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.
Dissipative Dynamics of Enzymes

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni; Zocchi LabMolecular Biophysics Team

2015-03-01

We explore enzyme conformational dynamics at sub - Å resolution, specifically temperature effects. The ensemble averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor 2 as the temperature is raised from 10 C to 45 C; the elastic parameter K shows a similar decrease. Thus when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.
Dissipative dynamics of enzymes.

PubMed

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

2014-11-07

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.
Regulation of C:N:P stoichiometry of microbes and soil organic matter by optimizing enzyme allocation: an omics-informed model study

NASA Astrophysics Data System (ADS)

Song, Y.; Yao, Q.; Wang, G.; Yang, X.; Mayes, M. A.

2017-12-01

Increasing evidences is indicating that soil organic matter (SOM) decomposition and stabilization process is a continuum process and controlled by both microbial functions and their interaction with minerals (known as the microbial efficiency-matrix stabilization theory (MEMS)). Our metagenomics analysis of soil samples from both P-deficit and P-fertilization sites in Panama has demonstrated that community-level enzyme functions could adapt to maximize the acquisition of limiting nutrients and minimize energy demand for foraging (known as the optimal foraging theory). This optimization scheme can mitigate the imbalance of C/P ratio between soil substrate and microbial community and relieve the P limitation on microbial carbon use efficiency over the time. Dynamic allocation of multiple enzyme groups and their interaction with microbial/substrate stoichiometry has rarely been considered in biogeochemical models due to the difficulties in identifying microbial functional groups and quantifying the change in enzyme expression in response to soil nutrient availability. This study aims to represent the omics-informed optimal foraging theory in the Continuum Microbial ENzyme Decomposition model (CoMEND), which was developed to represent the continuum SOM decomposition process following the MEMS theory. The SOM pools in the model are classified based on soil chemical composition (i.e. Carbohydrates, lignin, N-rich SOM and P-rich SOM) and the degree of SOM depolymerization. The enzyme functional groups for decomposition of each SOM pool and N/P mineralization are identified by the relative composition of gene copy numbers. The responses of microbial activities and SOM decomposition to nutrient availability are simulated by optimizing the allocation of enzyme functional groups following the optimal foraging theory. The modeled dynamic enzyme allocation in response to P availability is evaluated by the metagenomics data measured from P addition and P-deficit soil samples in Panama sites.The implementation of dynamic enzyme allocation in response to nutrient availability in the CoMEND model enables us to capture the varying microbial C/P ratio and soil carbon dynamics in response to shifting nutrient constraints over time in tropical soils.
Functional Enzyme-Based Approach for Linking Microbial Community Functions with Biogeochemical Process Kinetics

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

Li, Minjing; Qian, Wei-jun; Gao, Yuqian

The kinetics of biogeochemical processes in natural and engineered environmental systems are typically described using Monod-type or modified Monod-type models. These models rely on biomass as surrogates for functional enzymes in microbial community that catalyze biogeochemical reactions. A major challenge to apply such models is the difficulty to quantitatively measure functional biomass for constraining and validating the models. On the other hand, omics-based approaches have been increasingly used to characterize microbial community structure, functions, and metabolites. Here we proposed an enzyme-based model that can incorporate omics-data to link microbial community functions with biogeochemical process kinetics. The model treats enzymes asmore » time-variable catalysts for biogeochemical reactions and applies biogeochemical reaction network to incorporate intermediate metabolites. The sequences of genes and proteins from metagenomes, as well as those from the UniProt database, were used for targeted enzyme quantification and to provide insights into the dynamic linkage among functional genes, enzymes, and metabolites that are necessary to be incorporated in the model. The application of the model was demonstrated using denitrification as an example by comparing model-simulated with measured functional enzymes, genes, denitrification substrates and intermediates« less
In silico strategies toward enzyme function and dynamics.

PubMed

Estácio, Sílvia G

2012-01-01

Enzymes are outstanding biocatalysts involved in a plethora of chemical reactions occurring in the cell. Despite their incommensurable importance, a comprehensive understanding of enzyme catalysis is still missing. This task becomes more laborious given the unavoidability of including the inherent dynamic nature of enzymes into that description. As such, it is essential to ascertain the nature and contribution of enzyme conformational changes to catalysis and to evaluate the adequacy of the proposal associating protein internal motions to the rate enhancement achieved. Dynamic events in enzymes span a wide range of time- and length-scales which have led to a surge in multiscale methodologies targeting enzyme function and dynamics. Computational strategies assume a preponderant role in such studies by allowing the atomic detail investigation of the fundamental mechanisms of enzyme catalysis thus surpassing what is achievable through experiments. While high-accuracy quantum mechanical methods are indicated to uncover the details of the chemical reaction occurring at the active site, molecular mechanical force fields and molecular dynamics approaches provide powerful means to access the conformational energy landscape accessible to enzymes. This review outlines some of the most important in silico methodologies in this area, highlighting examples of problems tackled and the insights obtained. Copyright © 2012 Elsevier Inc. All rights reserved.
Image registration and analysis for quantitative myocardial perfusion: application to dynamic circular cardiac CT.

PubMed

Isola, A A; Schmitt, H; van Stevendaal, U; Begemann, P G; Coulon, P; Boussel, L; Grass, M

2011-09-21

Large area detector computed tomography systems with fast rotating gantries enable volumetric dynamic cardiac perfusion studies. Prospectively, ECG-triggered acquisitions limit the data acquisition to a predefined cardiac phase and thereby reduce x-ray dose and limit motion artefacts. Even in the case of highly accurate prospective triggering and stable heart rate, spatial misalignment of the cardiac volumes acquired and reconstructed per cardiac cycle may occur due to small motion pattern variations from cycle to cycle. These misalignments reduce the accuracy of the quantitative analysis of myocardial perfusion parameters on a per voxel basis. An image-based solution to this problem is elastic 3D image registration of dynamic volume sequences with variable contrast, as it is introduced in this contribution. After circular cone-beam CT reconstruction of cardiac volumes covering large areas of the myocardial tissue, the complete series is aligned with respect to a chosen reference volume. The results of the registration process and the perfusion analysis with and without registration are evaluated quantitatively in this paper. The spatial alignment leads to improved quantification of myocardial perfusion for three different pig data sets.
Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.
Modeling extracellular matrix (ECM) alterations in ovarian cancer by multiphoton excited fabrication of stromal models (Conference Presentation)

NASA Astrophysics Data System (ADS)

Campagnola, Paul J.; Ajeti, Visar; Lara, Jorge; Eliceiri, Kevin W.; Patankar, Mansh

2016-04-01

A profound remodeling of the extracellular matrix (ECM) occurs in human ovarian cancer but it unknown how this affects tumor growth, where this understanding could lead to better diagnostics and therapeutic approaches. We investigate the role of these ECM alterations by using multiphoton excited (MPE) polymerization to fabricate biomimetic models to investigate operative cell-matrix interactions in invasion/metastasis. First, we create nano/microstructured gradients mimicking the basal lamina to study adhesion/migration dynamics of ovarian cancer cells of differing metastatic potential. We find a strong haptotactic response that depends on both contact guidance and ECM binding cues. While we found enhanced migration for more invasive cells, the specifics of alignment and directed migration also depend on cell polarity. We further use MPE fabrication to create collagen scaffolds with complex, 3D submicron morphology. The stromal scaffold designs are derived directly from "blueprints" based on SHG images of normal, high risk, and malignant ovarian tissues. The models are seeded with different cancer cell lines and this allows decoupling of the roles of cell characteristics (metastatic potential) and ECM structure and composition (normal vs cancer) on adhesion/migration dynamics. We found the malignant stroma structure promotes enhanced migration and proliferation and also cytoskeletal alignment. Creating synthetic models based on fibers patterns further allows decoupling the topographic roles of the fibers themselves vs their alignment within the tissue. These models cannot be synthesized by other conventional fabrication methods and we suggest the MPE image-based fabrication method will enable a variety of studies in cancer biology.
Spontaneous flow in polar active fluids: the effect of a phenomenological self propulsion-like term.

PubMed

Bonelli, Francesco; Gonnella, Giuseppe; Tiribocchi, Adriano; Marenduzzo, Davide

2016-01-01

We present hybrid lattice Boltzmann simulations of extensile and contractile active fluids where we incorporate phenomenologically the tendency of active particles such as cell and bacteria, to move, or swim, along the local orientation. Quite surprisingly, we show that the interplay between alignment and activity can lead to completely different results, according to geometry (periodic boundary conditions or confinement between flat walls) and nature of the activity (extensile or contractile). An interesting generic outcome is that the alignment interaction can transform stationary active patterns into continuously moving ones: the dynamics of these evolving patterns can be oscillatory or chaotic according to the strength of the alignment term. Our results suggest that flow-polarisation alignment can have important consequences on the collective dynamics of active fluids and active gel.
Polymer chain alignment and transistor properties of nanochannel-templated poly(3-hexylthiophene) nanowires

NASA Astrophysics Data System (ADS)

Oh, Seungjun; Hayakawa, Ryoma; Pan, Chengjun; Sugiyasu, Kazunori; Wakayama, Yutaka

2016-08-01

Nanowires of semiconducting poly(3-hexylthiophene) (P3HT) were produced by a nanochannel-template technique. Polymer chain alignment in P3HT nanowires was investigated as a function of nanochannel widths (W) and polymer chain lengths (L). We found that the ratio between chain length and channel width (L/W) was a key parameter as regards promoting polymer chain alignment. Clear dichroism was observed in polarized ultraviolet-visible (UV-Vis) absorption spectra only at a ratio of approximately L/W = 2, indicating that the L/W ratio must be optimized to achieve uniaxial chain alignment in the nanochannel direction. We speculate that an appropriate L/W ratio is effective in confining the geometries and conformations of polymer chains. This discussion was supported by theoretical simulations based on molecular dynamics. That is, the geometry of the polymer chains, including the distance and tilting angles of the chains in relation to the nanochannel surface, was dominant in determining the longitudinal alignment along the nanochannels. Thus prepared highly aligned polymer nanowire is advantageous for electrical carrier transport and has great potential for improving the device performance of field-effect transistors. In fact, a one-order improvement in carrier mobility was observed in a P3HT nanowire transistor.
Relationship between nanotopographical alignment and stem cell fate with live imaging and shape analysis

NASA Astrophysics Data System (ADS)

Newman, Peter; Galenano-Niño, Jorge Luis; Graney, Pamela; Razal, Joselito M.; Minett, Andrew I.; Ribas, João; Ovalle-Robles, Raquel; Biro, Maté; Zreiqat, Hala

2016-12-01

The topography of a biomaterial regulates cellular interactions and determine stem cell fate. A complete understanding of how topographical properties affect cell behavior will allow the rational design of material surfaces that elicit specified biological functions once placed in the body. To this end, we fabricate substrates with aligned or randomly organized fibrous nanostructured topographies. Culturing adipose-derived stem cells (ASCs), we explore the dynamic relationship between the alignment of topography, cell shape and cell differentiation to osteogenic and myogenic lineages. We show aligned topographies differentiate cells towards a satellite cell muscle progenitor state - a distinct cell myogenic lineage responsible for postnatal growth and repair of muscle. We analyze cell shape between the different topographies, using fluorescent time-lapse imaging over 21 days. In contrast to previous work, this allows the direct measurement of cell shape at a given time rather than defining the morphology of the underlying topography and neglecting cell shape. We report quantitative metrics of the time-based morphological behaviors of cell shape in response to differing topographies. This analysis offers insights into the relationship between topography, cell shape and cell differentiation. Cells differentiating towards a myogenic fate on aligned topographies adopt a characteristic elongated shape as well as the alignment of cells.
Exploring Protein Structure and Dynamics through a Project-Oriented Biochemistry Laboratory Module

ERIC Educational Resources Information Center

Lipchock, James M.; Ginther, Patrick S.; Douglas, Bonnie B.; Bird, Kelly E.; Loria, J. Patrick

2017-01-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant…
Structural studies of viperin, an antiviral radical SAM enzyme.

PubMed

Fenwick, Michael K; Li, Yue; Cresswell, Peter; Modis, Yorgo; Ealick, Steven E

2017-06-27

Viperin is an IFN-inducible radical S -adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S -adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα) 6 -barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.
Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

PubMed

Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A L

2015-08-01

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. © 2015 American Society of Plant Biologists. All Rights Reserved.
Intermediate Greek EFL Learners' Attitudes to On-Line Teaching Practices: A Blended Task-Based English Language Learning Approach

ERIC Educational Resources Information Center

Liontou, Trisevgeni

2015-01-01

This paper reports on a one-year longitudinal study that adopted a blended teaching approach based on designing and implementing an online EFL course to be used by Greek students aged 13-14 years old along their more traditional face-to-face lessons. The reason for creating a more dynamic learning environment aligned with the rest of the…
Growth of Vertically Aligned ZnO Nanowire Arrays Using Bilayered Metal Catalysts

DTIC Science & Technology

2012-01-01

12] J. P. Liu, C. X. Guo, C. M. Li et al., “Carbon-decorated ZnO nanowire array: a novel platform for direct electrochemistry of enzymes and...cited. Vertically aligned, high-density ZnO nanowires (NWs) were grown for the first time on c-plane sapphire using binary alloys of Ni/Au or Cu/Au as...deleterious to the ZnO NW array growth. Significant improvement of the Au adhesion on the substrate was noted, opening the potential for direct
Individual responses to alignment perturbations in socket reaction moments while walking in transtibial prostheses.

PubMed

Kobayashi, Toshiki; Orendurff, Michael S; Zhang, Ming; Boone, David A

2014-05-01

The alignment of transtibial prostheses has a systematic effect on the mean socket reaction moments in amputees. However, understanding their individual differences in response to alignment perturbations is also important for prosthetists to fully utilize the socket reaction moments for dynamic alignment in each unique patient. The aim of this study was to investigate individual responses to alignment perturbations in transtibial prostheses with solid-ankle-cushion-heel feet. A custom instrumented prosthesis alignment component was used to measure the socket reaction moments while walking in 11 amputees with transtibial prostheses under 17 alignment conditions, including 3° and 6° of flexion, extension, abduction, and adduction of the socket, 5mm and 10mm of anterior, posterior, lateral, and medial translation of the socket, and an initial baseline alignment. Coronal moments at 30% of stance and maximum sagittal moments were extracted for comparisons from each amputee. In the coronal plane, varus moment at 30% of stance was generally reduced by adduction or medial translation of the socket in all the amputees. In the sagittal plane, extension moment was generally increased by posterior translation or flexion of the socket; however, this was not necessarily the case for all the amputees. Individual responses to alignment perturbations are not always consistent, and prosthetists would need to be aware of this variance when addressing individual socket reaction moments during dynamic alignment in clinical setting. Copyright © 2014 Elsevier Ltd. All rights reserved.
Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus

PubMed Central

Safary, Azam; Moniri, Rezvan; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

2016-01-01

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries. PMID:28101462
Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus.

PubMed

Safary, Azam; Moniri, Rezvan; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

2016-12-01

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.
Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites.

PubMed

Kim, Hojin; Zhu, Bohan; Chen, Huiying; Adetiba, Oluwatomiyin; Agrawal, Aditya; Ajayan, Pulickel; Jacot, Jeffrey G; Verduzco, Rafael

2016-02-06

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs - polysiloxane-based and epoxy-based - are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 °C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.
Dynamic Knee Alignment and Collateral Knee Laxity and Its Variations in Normal Humans

PubMed Central

Deep, Kamal; Picard, Frederic; Clarke, Jon V.

2015-01-01

Alignment of normal, arthritic, and replaced human knees is a much debated subject as is the collateral ligamentous laxity. Traditional quantitative values have been challenged. Methods used to measure these are also not without flaws. Authors review the recent literature and a novel method of measurement of these values has been included. This method includes use of computer navigation technique in clinic setting for assessment of the normal or affected knee before the surgery. Computer navigation has been known for achievement of alignment accuracy during knee surgery. Now its use in clinic setting has added to the inventory of measurement methods. Authors dispel the common myth of straight mechanical axis in normal knees and also look at quantification of amount of collateral knee laxity. Based on the scientific studies, it has been shown that the mean alignment is in varus in normal knees. It changes from lying non-weight-bearing position to standing weight-bearing position in both coronal and the sagittal planes. It also varies with gender and race. The collateral laxity is also different for males and females. Further studies are needed to define the ideal alignment and collateral laxity which the surgeon should aim for individual knees. PMID:26636090
Identification of chikungunya virus nsP2 protease inhibitors using structure-base approaches.

PubMed

Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

2015-04-01

The nsP2 protease of chikungunya virus (CHIKV) is one of the essential components of viral replication and it plays a crucial role in the cleavage of polyprotein precursors for the viral replication process. Therefore, it is gaining attention as a potential drug design target against CHIKV. Based on the recently determined crystal structure of the nsP2 protease of CHIKV, this study identified potential inhibitors of the virus using structure-based approaches with a combination of molecular docking, virtual screening and molecular dynamics (MD) simulations. The top hit compounds from database searching, using the NCI Diversity Set II, with targeting at five potential binding sites of the nsP2 protease, were identified by blind dockings and focused dockings. These complexes were then subjected to MD simulations to investigate the stability and flexibility of the complexes and to gain a more detailed insight into the interactions between the compounds and the enzyme. The hydrogen bonds and hydrophobic contacts were characterized for the complexes. Through structural alignment, the catalytic residues Cys1013 and His1083 were identified in the N-terminal region of the nsP2 protease. The absolute binding free energies were estimated by the linear interaction energy approach and compared with the binding affinities predicted with docking. The results provide valuable information for the development of inhibitors for CHIKV. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.
First-Principles Approach to Energy Level Alignment at Aqueous Semiconductor Interfaces

NASA Astrophysics Data System (ADS)

Hybertsen, Mark

2015-03-01

We have developed a first principles method to calculate the energy level alignment between semiconductor band edges and reference energy levels at aqueous interfaces. This alignment is fundamental to understand the electrochemical characteristics of any semiconductor electrode in general and the potential for photocatalytic activity in particular. For example, in the search for new photo-catalytic materials, viable candidates must demonstrate both efficient absorption of the solar spectrum and an appropriate alignment of the band edge levels in the semiconductor to the redox levels for the target reactions. In our approach, the interface-specific contribution to the electrostatic step across the interface is evaluated using density functional theory (DFT) based molecular dynamics to sample the physical interface structure and the corresponding change in the electrostatic potential at the interface. The reference electronic levels in the semiconductor and in the water are calculated using the GW approach, which naturally corrects for errors inherent in the use of Kohn-Sham energy eigenvalues to approximate the electronic excitation energies in each material. Taken together, our calculations provide the alignment of the semiconductor valence band edge to the centroid of the highest occupied 1b1 level in water. The known relationship of the 1b1 level to the normal hydrogen electrode completes the connection to electrochemical levels. We discuss specific results for GaN, ZnO, and TiO2. The effect of interface structural motifs, such as different degrees of water dissociation, and of dynamical characteristics, will be presented together with available experimental data. Work supported by the US Department of Energy, Office of Basic Energy Sciences under Contract No. DE-AC02-98CH10886.
Transcription Factor Map Alignment of Promoter Regions

PubMed Central

Blanco, Enrique; Messeguer, Xavier; Smith, Temple F; Guigó, Roderic

2006-01-01

We address the problem of comparing and characterizing the promoter regions of genes with similar expression patterns. This remains a challenging problem in sequence analysis, because often the promoter regions of co-expressed genes do not show discernible sequence conservation. In our approach, thus, we have not directly compared the nucleotide sequence of promoters. Instead, we have obtained predictions of transcription factor binding sites, annotated the predicted sites with the labels of the corresponding binding factors, and aligned the resulting sequences of labels—to which we refer here as transcription factor maps (TF-maps). To obtain the global pairwise alignment of two TF-maps, we have adapted an algorithm initially developed to align restriction enzyme maps. We have optimized the parameters of the algorithm in a small, but well-curated, collection of human–mouse orthologous gene pairs. Results in this dataset, as well as in an independent much larger dataset from the CISRED database, indicate that TF-map alignments are able to uncover conserved regulatory elements, which cannot be detected by the typical sequence alignments. PMID:16733547
Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily.

PubMed

Narayanan, Chitra; Bernard, David N; Bafna, Khushboo; Gagné, Donald; Chennubhotla, Chakra S; Doucet, Nicolas; Agarwal, Pratul K

2018-03-06

Enzyme superfamily members that share common chemical and/or biological functions also share common features. While the role of structure is well characterized, the link between enzyme function and dynamics is not well understood. We present a systematic characterization of intrinsic dynamics of over 20 members of the pancreatic-type RNase superfamily, which share a common structural fold. This study is motivated by the fact that the range of chemical activity as well as molecular motions of RNase homologs spans over 10 5 folds. Dynamics was characterized using a combination of nuclear magnetic resonance experiments and computer simulations. Phylogenetic clustering led to the grouping of sequences into functionally distinct subfamilies. Detailed characterization of the diverse RNases showed conserved dynamical traits for enzymes within subfamilies. These results suggest that selective pressure for the conservation of dynamical behavior, among other factors, may be linked to the distinct chemical and biological functions in an enzyme superfamily. Copyright © 2018 Elsevier Ltd. All rights reserved.
Dynamic Tensile Loading Improves the Functional Properties of Mesenchymal Stem Cell-Laden Nanofiber-Based Fibrocartilage

PubMed Central

Baker, Brendon M.; Shah, Roshan P.; Huang, Alice H.

2011-01-01

Fibrocartilaginous tissues such as the meniscus serve critical load-bearing roles, relying on arrays of collagen fibers to resist tensile loads experienced with normal activity. As these structures are frequently injured and possess limited healing capacity, there exists great demand for tissue-engineered replacements. Toward recreating the structural features of these anisotropic tissues in vitro, we employ scaffolds composed of co-aligned nanofibers that direct mesenchymal stem cell (MSC) orientation and the formation of organized extracellular matrix (ECM). Concomitant with ECM synthesis, the mechanical properties of constructs increase with free-swelling culture, but ultimately failed to achieve equivalence with meniscal fibrocartilage. As mechanical forces are essential to the development and maintenance of musculoskeletal tissues, this work examined the effect of cyclic tensile loading on MSC-laden nanofibrous constructs. We hypothesized that loading would modulate the transcriptional behavior of MSCs, spur the deposition of ECM, and lead to enhancements in construct mechanical properties compared to free-swelling controls. Fiber-aligned scaffolds were seeded with MSCs and dynamically loaded daily in tension or maintained as nonloaded controls for 4 weeks. With mechanical stimulation, fibrous gene expression increased, collagen deposition increased, and the tensile modulus increased by 16% relative to controls. These results show that dynamic tensile loading enhances the maturation of MSC-laden aligned nanofibrous constructs, suggesting that recapitulation of the structural and mechanical environment of load-bearing tissues results in increases in functional properties that can be exploited for tissue engineering applications. PMID:21247342
Dynamic tensile loading improves the functional properties of mesenchymal stem cell-laden nanofiber-based fibrocartilage.

PubMed

Baker, Brendon M; Shah, Roshan P; Huang, Alice H; Mauck, Robert L

2011-05-01

Fibrocartilaginous tissues such as the meniscus serve critical load-bearing roles, relying on arrays of collagen fibers to resist tensile loads experienced with normal activity. As these structures are frequently injured and possess limited healing capacity, there exists great demand for tissue-engineered replacements. Toward recreating the structural features of these anisotropic tissues in vitro, we employ scaffolds composed of co-aligned nanofibers that direct mesenchymal stem cell (MSC) orientation and the formation of organized extracellular matrix (ECM). Concomitant with ECM synthesis, the mechanical properties of constructs increase with free-swelling culture, but ultimately failed to achieve equivalence with meniscal fibrocartilage. As mechanical forces are essential to the development and maintenance of musculoskeletal tissues, this work examined the effect of cyclic tensile loading on MSC-laden nanofibrous constructs. We hypothesized that loading would modulate the transcriptional behavior of MSCs, spur the deposition of ECM, and lead to enhancements in construct mechanical properties compared to free-swelling controls. Fiber-aligned scaffolds were seeded with MSCs and dynamically loaded daily in tension or maintained as nonloaded controls for 4 weeks. With mechanical stimulation, fibrous gene expression increased, collagen deposition increased, and the tensile modulus increased by 16% relative to controls. These results show that dynamic tensile loading enhances the maturation of MSC-laden aligned nanofibrous constructs, suggesting that recapitulation of the structural and mechanical environment of load-bearing tissues results in increases in functional properties that can be exploited for tissue engineering applications.
Matt: local flexibility aids protein multiple structure alignment.

PubMed

Menke, Matthew; Berger, Bonnie; Cowen, Lenore

2008-01-01

Even when there is agreement on what measure a protein multiple structure alignment should be optimizing, finding the optimal alignment is computationally prohibitive. One approach used by many previous methods is aligned fragment pair chaining, where short structural fragments from all the proteins are aligned against each other optimally, and the final alignment chains these together in geometrically consistent ways. Ye and Godzik have recently suggested that adding geometric flexibility may help better model protein structures in a variety of contexts. We introduce the program Matt (Multiple Alignment with Translations and Twists), an aligned fragment pair chaining algorithm that, in intermediate steps, allows local flexibility between fragments: small translations and rotations are temporarily allowed to bring sets of aligned fragments closer, even if they are physically impossible under rigid body transformations. After a dynamic programming assembly guided by these "bent" alignments, geometric consistency is restored in the final step before the alignment is output. Matt is tested against other recent multiple protein structure alignment programs on the popular Homstrad and SABmark benchmark datasets. Matt's global performance is competitive with the other programs on Homstrad, but outperforms the other programs on SABmark, a benchmark of multiple structure alignments of proteins with more distant homology. On both datasets, Matt demonstrates an ability to better align the ends of alpha-helices and beta-strands, an important characteristic of any structure alignment program intended to help construct a structural template library for threading approaches to the inverse protein-folding problem. The related question of whether Matt alignments can be used to distinguish distantly homologous structure pairs from pairs of proteins that are not homologous is also considered. For this purpose, a p-value score based on the length of the common core and average root mean squared deviation (RMSD) of Matt alignments is shown to largely separate decoys from homologous protein structures in the SABmark benchmark dataset. We postulate that Matt's strong performance comes from its ability to model proteins in different conformational states and, perhaps even more important, its ability to model backbone distortions in more distantly related proteins.
Dynamic trunk stabilization: a conceptual back injury prevention program for volleyball athletes.

PubMed

Smith, Chad E; Nyland, John; Caudill, Paul; Brosky, Joseph; Caborn, David N M

2008-11-01

The sport of volleyball creates considerable dynamic trunk stability demands. Back injury occurs all too frequently in volleyball, particularly among female athletes. The purpose of this clinical commentary is to review functional anatomy, muscle coactivation strategies, assessment of trunk muscle performance, and the characteristics of effective exercises for the trunk or core. From this information, a conceptual progressive 3-phase volleyball-specific training program is presented to improve dynamic trunk stability and to potentially reduce the incidence of back injury among volleyball athletes. Phase 1 addresses low-velocity motor control, kinesthetic awareness, and endurance, with the clinician providing cues to teach achievement of biomechanically neutral spine alignment. Phase 2 focuses on progressively higher velocity dynamic multiplanar endurance, coordination, and strength-power challenges integrating upper and lower extremity movements, while maintaining neutral spine alignment. Phase 3 integrates volleyball-specific skill simulations by breaking down composite movement patterns into their component parts, with differing dynamic trunk stability requirements, while maintaining neutral spine alignment. Prospective research is needed to validate the efficacy of this program.
A short note on dynamic programming in a band.

PubMed

Gibrat, Jean-François

2018-06-15

Third generation sequencing technologies generate long reads that exhibit high error rates, in particular for insertions and deletions which are usually the most difficult errors to cope with. The only exact algorithm capable of aligning sequences with insertions and deletions is a dynamic programming algorithm. In this note, for the sake of efficiency, we consider dynamic programming in a band. We show how to choose the band width in function of the long reads' error rates, thus obtaining an [Formula: see text] algorithm in space and time. We also propose a procedure to decide whether this algorithm, when applied to semi-global alignments, provides the optimal score. We suggest that dynamic programming in a band is well suited to the problem of aligning long reads between themselves and can be used as a core component of methods for obtaining a consensus sequence from the long reads alone. The function implementing the dynamic programming algorithm in a band is available, as a standalone program, at: https://forgemia.inra.fr/jean-francois.gibrat/BAND_DYN_PROG.git.
Vertically aligned carbon nanotube probes for monitoring blood cholesterol

NASA Astrophysics Data System (ADS)

Roy, Somenath; Vedala, Harindra; Choi, Wonbong

2006-02-01

Detection of blood cholesterol is of great clinical significance. The amperometric detection technique was used for the enzymatic assay of total cholesterol. Multiwall carbon nanotubes (MWNTs), vertically aligned on a silicon platform, promote heterogeneous electron transfer between the enzyme and the working electrode. Surface modification of the MWNT with a biocompatible polymer, polyvinyl alcohol (PVA), converted the hydrophobic nanotube surface into a highly hydrophilic one, which facilitates efficient attachment of biomolecules. The fabricated working electrodes showed a linear relationship between cholesterol concentration and the output signal. The efficacy of the multiwall carbon nanotubes in promoting heterogeneous electron transfer was evident by distinct electrochemical peaks and higher signal-to-noise ratio as compared to the Au electrode with identical enzyme immobilization protocol. The selectivity of the cholesterol sensor in the presence of common interferents present in human blood, e.g. uric acid, ascorbic acid and glucose, is also reported.
Modified kinetics of enzymes interacting with nanoparticles

NASA Astrophysics Data System (ADS)

Díaz, Sebastián. A.; Breger, Joyce C.; Malanoski, Anthony; Claussen, Jonathan C.; Walper, Scott A.; Ancona, Mario G.; Brown, Carl W.; Stewart, Michael H.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

2015-08-01

Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.
Generic accelerated sequence alignment in SeqAn using vectorization and multi-threading.

PubMed

Rahn, René; Budach, Stefan; Costanza, Pascal; Ehrhardt, Marcel; Hancox, Jonny; Reinert, Knut

2018-05-03

Pairwise sequence alignment is undoubtedly a central tool in many bioinformatics analyses. In this paper, we present a generically accelerated module for pairwise sequence alignments applicable for a broad range of applications. In our module, we unified the standard dynamic programming kernel used for pairwise sequence alignments and extended it with a generalized inter-sequence vectorization layout, such that many alignments can be computed simultaneously by exploiting SIMD (Single Instruction Multiple Data) instructions of modern processors. We then extended the module by adding two layers of thread-level parallelization, where we a) distribute many independent alignments on multiple threads and b) inherently parallelize a single alignment computation using a work stealing approach producing a dynamic wavefront progressing along the minor diagonal. We evaluated our alignment vectorization and parallelization on different processors, including the newest Intel® Xeon® (Skylake) and Intel® Xeon Phi™ (KNL) processors, and use cases. The instruction set AVX512-BW (Byte and Word), available on Skylake processors, can genuinely improve the performance of vectorized alignments. We could run single alignments 1600 times faster on the Xeon Phi™ and 1400 times faster on the Xeon® than executing them with our previous sequential alignment module. The module is programmed in C++ using the SeqAn (Reinert et al., 2017) library and distributed with version 2.4. under the BSD license. We support SSE4, AVX2, AVX512 instructions and included UME::SIMD, a SIMD-instruction wrapper library, to extend our module for further instruction sets. We thoroughly test all alignment components with all major C++ compilers on various platforms. rene.rahn@fu-berlin.de.
Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.
Investigation of Rhodopsin Dynamics in its Signaling State by Solid-State Deuterium NMR Spectroscopy

PubMed Central

Struts, Andrey V.; Chawla, Udeep; Perera, Suchithranga M.D.C.; Brown, Michael F.

2017-01-01

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state 2H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. In addition, solid-state 2H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state 2H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition, we review optimized conditions for trapping the rhodopsin photointermediates; and lastly we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films. PMID:25697522
The kinetic regime of the Vicsek model

NASA Astrophysics Data System (ADS)

Chepizhko, A. A.; Kulinskii, V. L.

2009-12-01

We consider the dynamics of the system of self-propelling particles modeled via the Vicsek algorithm in continuum time limit. It is shown that the alignment process for the velocities can be subdivided into two regimes: "fast" kinetic and "slow" hydrodynamic ones. In fast kinetic regime the alignment of the particle velocity to the local neighborhood takes place with characteristic relaxation time. So, that the bigger regions arise with the velocity alignment. These regions align their velocities thus giving rise to hydrodynamic regime of the dynamics. We propose the mean-field-like approach in which we take into account the correlations between density and velocity. The comparison of the theoretical predictions with the numerical simulations is given. The relation between Vicsek model in the zero velocity limit and the Kuramoto model is stated. The mean-field approach accounting for the dynamic change of the neighborhood is proposed. The nature of the discontinuity of the dependence of the order parameter in case of vectorial noise revealed in Gregorie and Chaite, Phys. Rev. Lett., 92, 025702 (2004) is discussed and the explanation of it is proposed.
Vertically Aligned Carbon Nanofiber based Biosensor Platform for Glucose Sensor

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

Al Mamun, Khandaker A.; Tulip, Fahmida S.; MacArthur, Kimberly

2014-03-01

Vertically aligned carbon nanofibers (VACNFs) have recently become an important tool for biosensor design. Carbon nanofibers (CNF) have excellent conductive and structural properties with many irregularities and defect sites in addition to exposed carboxyl groups throughout their surfaces. These properties allow a better immobilization matrix compared to carbon nanotubes and offer better resolution when compared with the FET-based biosensors. VACNFs can be deterministically grown on silicon substrates allowing optimization of the structures for various biosensor applications. Two VACNF electrode architectures have been employed in this study and a comparison of their performances has been made in terms of sensitivity, sensingmore » limitations, dynamic range, and response time. The usage of VACNF platform as a glucose sensor has been verified in this study by selecting an optimum architecture based on the VACNF forest density. Read More: http://www.worldscientific.com/doi/abs/10.1142/S0129156414500062« less

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

PubMed Central

Luk, Louis Y. P.; Javier Ruiz-Pernía, J.; Dawson, William M.; Roca, Maite; Loveridge, E. Joel; Glowacki, David R.; Harvey, Jeremy N.; Mulholland, Adrian J.; Tuñón, Iñaki; Moliner, Vicent; Allemann, Rudolf K.

2013-01-01

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy (15N, 13C, 2H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for “promoting motions” in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate. PMID:24065822
Enzyme Sequestration as a Tuning Point in Controlling Response Dynamics of Signalling Networks

PubMed Central

Ollivier, Julien F.; Soyer, Orkun S.

2016-01-01

Signalling networks result from combinatorial interactions among many enzymes and scaffolding proteins. These complex systems generate response dynamics that are often essential for correct decision-making in cells. Uncovering biochemical design principles that underpin such response dynamics is a prerequisite to understand evolved signalling networks and to design synthetic ones. Here, we use in silico evolution to explore the possible biochemical design space for signalling networks displaying ultrasensitive and adaptive response dynamics. By running evolutionary simulations mimicking different biochemical scenarios, we find that enzyme sequestration emerges as a key mechanism for enabling such dynamics. Inspired by these findings, and to test the role of sequestration, we design a generic, minimalist model of a signalling cycle, featuring two enzymes and a single scaffolding protein. We show that this simple system is capable of displaying both ultrasensitive and adaptive response dynamics. Furthermore, we find that tuning the concentration or kinetics of the sequestering protein can shift system dynamics between these two response types. These empirical results suggest that enzyme sequestration through scaffolding proteins is exploited by evolution to generate diverse response dynamics in signalling networks and could provide an engineering point in synthetic biology applications. PMID:27163612
Dynamical differences of hemoglobin and the ionotropic glutamate receptor in different states revealed by a new dynamics alignment method.

PubMed

Tobi, Dror

2017-08-01

A new algorithm for comparison of protein dynamics is presented. Compared protein structures are superposed and their modes of motions are calculated using the anisotropic network model. The obtained modes are aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. Dynamical comparison of hemoglobin in the T and R2 states reveals that the dynamics of the allosteric effector 2,3-bisphosphoglycerate binding site is different in the two states. These differences can contribute to the selectivity of the effector to the T state. Similar comparison of the ionotropic glutamate receptor in the kainate+(R,R)-2b and ZK bound states reveals that the kainate+(R,R)-2b bound states slow modes describe upward motions of ligand binding domain and the transmembrane domain regions. Such motions may lead to the opening of the receptor. The upper lobes of the LBDs of the ZK bound state have a smaller interface with the amino terminal domains above them and have a better ability to move together. The present study exemplifies the use of dynamics comparison as a tool to study protein function. Proteins 2017; 85:1507-1517. © 2014 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
Structure-Based Virtual Screening and Biochemical Evaluation for the Identification of Novel Trypanosoma Brucei Aldolase Inhibitors.

PubMed

Ferreira, Leonardo L G; Ferreira, Rafaela S; Palomino, David L; Andricopulo, Adriano D

2018-04-27

The glycolytic enzyme fructose-1,6-bisphosphate aldolase is a validated molecular target in human African trypanosomiasis (HAT) drug discovery, a neglected tropical disease (NTD) caused by the protozoan Trypanosoma brucei. Herein, a structure-based virtual screening (SBVS) approach to the identification of novel T. brucei aldolase inhibitors is described. Distinct molecular docking algorithms were used to screen more than 500,000 compounds against the X-ray structure of the enzyme. This SBVS strategy led to the selection of a series of molecules which were evaluated for their activity on recombinant T. brucei aldolase. The effort led to the discovery of structurally new ligands able to inhibit the catalytic activity the enzyme. The predicted binding conformations were additionally investigated in molecular dynamics simulations, which provided useful insights into the enzyme-inhibitor intermolecular interactions. The molecular modeling results along with the enzyme inhibition data generated practical knowledge to be explored in further structure-based drug design efforts in HAT drug discovery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Molecular design of two sterol 14α-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole

NASA Astrophysics Data System (ADS)

Rupp, Bernd; Raub, Stephan; Marian, Christel; Höltje, Hans-Dieter

2005-03-01

Sterol 14α-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.
Organizational Strategy and Business Environment Effects Based on a Computation Method

NASA Astrophysics Data System (ADS)

Reklitis, Panagiotis; Konstantopoulos, Nikolaos; Trivellas, Panagiotis

2007-12-01

According to many researchers of organizational theory, a great number of problems encountered by the manufacturing firms are due to their ineffectiveness to respond to significant changes of their external environment and align their competitive strategy accordingly. From this point of view, the pursuit of the appropriate generic strategy is vital for firms facing a dynamic and highly competitive environment. In the present paper, we adopt Porter's typology to operationalise organizational strategy (cost leadership, innovative and marketing differentiation, and focus) considering changes in the external business environment (dynamism, complexity and munificence). Although simulation of social events is a quite difficult task, since there are so many considerations (not all well understood) involved, in the present study we developed a dynamic system based on the conceptual framework of strategy-environment associations.
A QM/MM Metadynamics Study of the Direct Decarboxylation Mechanism for Orotidine-5'-monophosphate Decarboxylase using Two Different QM Regions: Acceleration too Small to Explain Rate of Enzyme Catalysis

PubMed Central

Stanton, Courtney; Kuo, I-Feng W.; Mundy, Christopher J.; Laino, Teodoro; Houk, K. N.

2011-01-01

Despite decades of study, the mechanism by which orotidine-5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine monophosphate remains unresolved. A computational investigation of the direct decarboxylation mechanism has been performed using mixed quantum mechanical/molecular mechanical (QM/MM) dynamics simulations. The study was performed with the program CP2K that integrates classical dynamics and ab initio dynamics based on the Born-Oppenheimer approach. Two different QM regions were explored. The free energy barriers for decarboxylation of orotidine-5'-monophosphate (OMP) in solution and in the enzyme (using the larger QM region) were determined with the metadynamics method to be 40 kcal/mol and 33 kcal/mol, respectively. The calculated change in activation free energy (ΔΔG±) on going from solution to the enzyme is therefore −7 kcal/mol, far less than the experimental change of −23 kcal/mol (for kcat/kuncat Radzicka, A.; Wolfenden, R., Science. 1995, 267, 90–92). These results do not support the direct decarboxylation mechanism that has been proposed for the enzyme. However, in the context of QM/MM calculations, it was found that the size of the QM region has a dramatic effect on the calculated reaction barrier. PMID:17927240
The alignment of enzymatic steps reveals similar metabolic pathways and probable recruitment events in Gammaproteobacteria.

PubMed

Poot-Hernandez, Augusto Cesar; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

2015-11-17

It is generally accepted that gene duplication followed by functional divergence is one of the main sources of metabolic diversity. In this regard, there is an increasing interest in the development of methods that allow the systematic identification of these evolutionary events in metabolism. Here, we used a method not based on biomolecular sequence analysis to compare and identify common and variable routes in the metabolism of 40 Gammaproteobacteria species. The metabolic maps deposited in the KEGG database were transformed into linear Enzymatic Step Sequences (ESS) by using the breadth-first search algorithm. These ESS represent subsequent enzymes linked to each other, where their catalytic activities are encoded in the Enzyme Commission numbers. The ESS were compared in an all-against-all (pairwise comparisons) approach by using a dynamic programming algorithm, leaving only a set of significant pairs. From these comparisons, we identified a set of functionally conserved enzymatic steps in different metabolic maps, in which cell wall components and fatty acid and lysine biosynthesis were included. In addition, we found that pathways associated with biosynthesis share a higher proportion of similar ESS than degradation pathways and secondary metabolism pathways. Also, maps associated with the metabolism of similar compounds contain a high proportion of similar ESS, such as those maps from nucleotide metabolism pathways, in particular the inosine monophosphate pathway. Furthermore, diverse ESS associated with the low part of the glycolysis pathway were identified as functionally similar to multiple metabolic pathways. In summary, our comparisons may help to identify similar reactions in different metabolic pathways and could reinforce the patchwork model in the evolution of metabolism in Gammaproteobacteria.
Magnetically aligned phospholipid bilayers in weak magnetic fields: optimization, mechanism, and advantages for X-band EPR studies.

PubMed

Cardon, Thomas B; Tiburu, Elvis K; Lorigan, Gary A

2003-03-01

Our lab is developing a spin-labeled EPR spectroscopic technique complementary to solid-state NMR studies to study the structure, orientation, and dynamics of uniaxially aligned integral membrane proteins inserted into magnetically aligned discotic phospholipid bilayers, or bicelles. The focus of this study is to optimize and understand the mechanisms involved in the magnetic alignment process of bicelle disks in weak magnetic fields. Developing experimental conditions for optimized magnetic alignment of bicelles in low magnetic fields may prove useful to study the dynamics of membrane proteins and its interactions with lipids, drugs, steroids, signaling events, other proteins, etc. In weak magnetic fields, the magnetic alignment of Tm(3+)-doped bicelle disks was thermodynamically and kinetically very sensitive to experimental conditions. Tm(3+)-doped bicelles were magnetically aligned using the following optimized procedure: the temperature was slowly raised at a rate of 1.9K/min from an initial temperature being between 298 and 307K to a final temperature of 318K in the presence of a static magnetic field of 6300G. The spin probe 3beta-doxyl-5alpha-cholestane (cholestane) was inserted into the bicelle disks and utilized to monitor bicelle alignment by analyzing the anisotropic hyperfine splitting for the corresponding EPR spectra. The phases of the bicelles were determined using solid-state 2H NMR spectroscopy and compared with the corresponding EPR spectra. Macroscopic alignment commenced in the liquid crystalline nematic phase (307K), continued to increase upon slowly raising the temperature, and was well-aligned in the liquid crystalline lamellar smectic phase (318K).
A novel mechanism important for the alignment of microtubules.

PubMed

Wightman, Raymond; Turner, Simon R

2008-04-01

Using a live-cell imaging approach to study individual micro-tubules, we have compared microtubule behavior between net-like and aligned cortical arrays. In contrast to previous studies, a steep angled collision between the growing end of a microtubule and a preexisting microtubule was found to favor crossover. Frequencies of microtubule crossovers, bundling and catastrophes are similar regardless of whether the cell exhibited a net-like or aligned microtubule array. In the predominantly aligned array of petiole cells, severing occurs at the sites of microtubule crossovers and serves to remove unaligned microtubules and to increase microtubule density. Severing was observed to be rare in net-like arrays. Microtubule severing is carried out by the katanin enzyme. In this addendum, we present new insights into the possible mechanism of crossing over and preliminary data looking at organization of the array in a katanin mutant.
Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package

PubMed Central

Kumar, Yadhu; Westram, Ralf; Kipfer, Peter; Meier, Harald; Ludwig, Wolfgang

2006-01-01

Background Availability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA) for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment. Results Three-dimensional structure of rRNA is visualized in OpenGL 3D environment with the abilities to change the display and overlay information onto the molecule, dynamically. Phylogenetic information derived from the multiple sequence alignments can be overlaid onto the molecule structure in a real time. Superimposition of both statistical and non-statistical sequence associated information onto the rRNA 3D structure can be done using customizable color scheme, which is also applied to a textual sequence alignment for reference. Oligonucleotide probes designed by ARB probe design tools can be mapped onto the 3D structure along with the probe accessibility models for evaluation with respect to secondary and tertiary structural conformations of rRNA. Conclusion Visualization of three-dimensional structure of rRNA in an intuitive display provides the biologists with the greater possibilities to carry out structure based phylogenetic analysis. Coupled with secondary structure models of rRNA, RNA3D program aids in validating the sequence alignments of rRNA genes and evaluating probe target sites. Superimposition of the information derived from the multiple sequence alignment onto the molecule dynamically allows the researchers to observe any sequence inherited characteristics (phylogenetic information) in real-time environment. The extended ARB software package is made freely available for the scientific community via . PMID:16672074
Effect of electric field induced alignment and dispersion of functionalized carbon nanotubes on properties of natural rubber

NASA Astrophysics Data System (ADS)

Gao, Jiangshan; He, Yan; Gong, Xiubin

2018-06-01

The original equipment and method for orienting multi-walled carbon nanotubes (MWCNTs) in natural rubber (NR) by alternating current (AC) electric field were reported in the present study. MWCNTs with various volume fractions were dispersed in the mixture latex which composed of natural rubber, additives and methylbenzene. The application of AC electric field during nanocomposites curing process was used to induce the formation of aligned conductive nanotube networks between the electrodes. The aligned MWCNTs in the composites have a better orientation performance and dispersion quality than these of random MWCNTs by analyzing TEM and SEM images. The effects of MWCNTs anisotropy on thermal conductivity, dielectric properties, and dynamic mechanical properties of NR were studied. The mean value of thermal conductivity of composites loading with aligned MWCNTs was 8.67% higher than that of composites with random MWCNTs due to the anisotropy of aligned MWCNTs. The compounds with aligned MWCNTs possessed low dielectric constant, loss tangents and conductivity, namely a good insulativity. The compounds loading with aligned MWCNTs had lower loss modulus and better dynamic mechanical properties than those with random MWCNTs. This method can make full use of the high thermal conductivity of MWCNTs axis, and expand the application areas of natural rubber like conducting heat in a certain direction with a high efficiency.
Experience from the in-flight calibration of the Extreme Ultraviolet Explorer (EUVE) and Upper Atmosphere Research Satellite (UARS) fixed head star trackers (FHSTs)

NASA Technical Reports Server (NTRS)

Lee, Michael

1995-01-01

Since the original post-launch calibration of the FHSTs (Fixed Head Star Trackers) on EUVE (Extreme Ultraviolet Explorer) and UARS (Upper Atmosphere Research Satellite), the Flight Dynamics task has continued to analyze the FHST performance. The algorithm used for inflight alignment of spacecraft sensors is described and the equations for the errors in the relative alignment for the simple 2 star tracker case are shown. Simulated data and real data are used to compute the covariance of the relative alignment errors. Several methods for correcting the alignment are compared and results analyzed. The specific problems seen on orbit with UARS and EUVE are then discussed. UARS has experienced anomalous tracker performance on an FHST resulting in continuous variation in apparent tracker alignment. On EUVE, the FHST residuals from the attitude determination algorithm showed a dependence on the direction of roll during survey mode. This dependence is traced back to time tagging errors and the original post launch alignment is found to be in error due to the impact of the time tagging errors on the alignment algorithm. The methods used by the FDF (Flight Dynamics Facility) to correct for these problems is described.
The relative phases of basal ganglia activities dynamically shape effective connectivity in Parkinson's disease.

PubMed

Cagnan, Hayriye; Duff, Eugene Paul; Brown, Peter

2015-06-01

Optimal phase alignment between oscillatory neural circuits is hypothesized to optimize information flow and enhance system performance. This theory is known as communication-through-coherence. The basal ganglia motor circuit exhibits exaggerated oscillatory and coherent activity patterns in Parkinson's disease. Such activity patterns are linked to compromised motor system performance as evinced by bradykinesia, rigidity and tremor, suggesting that network function might actually deteriorate once a certain level of net synchrony is exceeded in the motor circuit. Here, we characterize the processes underscoring excessive synchronization and its termination. To this end, we analysed local field potential recordings from the subthalamic nucleus and globus pallidus of five patients with Parkinson's disease (four male and one female, aged 37-64 years). We observed that certain phase alignments between subthalamic nucleus and globus pallidus amplified local neural synchrony in the beta frequency band while others either suppressed it or did not induce any significant change with respect to surrogates. The increase in local beta synchrony directly correlated with how long the two nuclei locked to beta-amplifying phase alignments. Crucially, administration of the dopamine prodrug, levodopa, reduced the frequency and duration of periods during which subthalamic and pallidal populations were phase-locked to beta-amplifying alignments. Conversely ON dopamine, the total duration over which subthalamic and pallidal populations were aligned to phases that left beta-amplitude unchanged with respect to surrogates increased. Thus dopaminergic input shifted circuit dynamics from persistent periods of locking to amplifying phase alignments, associated with compromised motoric function, to more dynamic phase alignment and improved motoric function. This effect of dopamine on local circuit resonance suggests means by which novel electrical interventions might prevent resonance-related pathological circuit interactions. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.
Role of Dynamics in Enzyme Catalysis: Substantial versus Semantic Controversies

PubMed Central

2015-01-01

Conspectus The role of the enzyme’s dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term “protein dynamics” is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is “stored” in a specific protein mode and “used” for catalysis before it can dissipate to its environment (i.e., “nonstatistical dynamics”). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual “induced-fit” shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction’s transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions. PMID:25539442
Alignment-Independent Comparisons of Human Gastrointestinal Tract Microbial Communities in a Multidimensional 16S rRNA Gene Evolutionary Space▿

PubMed Central

Rudi, Knut; Zimonja, Monika; Kvenshagen, Bente; Rugtveit, Jarle; Midtvedt, Tore; Eggesbø, Merete

2007-01-01

We present a novel approach for comparing 16S rRNA gene clone libraries that is independent of both DNA sequence alignment and definition of bacterial phylogroups. These steps are the major bottlenecks in current microbial comparative analyses. We used direct comparisons of taxon density distributions in an absolute evolutionary coordinate space. The coordinate space was generated by using alignment-independent bilinear multivariate modeling. Statistical analyses for clone library comparisons were based on multivariate analysis of variance, partial least-squares regression, and permutations. Clone libraries from both adult and infant gastrointestinal tract microbial communities were used as biological models. We reanalyzed a library consisting of 11,831 clones covering complete colons from three healthy adults in addition to a smaller 390-clone library from infant feces. We show that it is possible to extract detailed information about microbial community structures using our alignment-independent method. Our density distribution analysis is also very efficient with respect to computer operation time, meeting the future requirements of large-scale screenings to understand the diversity and dynamics of microbial communities. PMID:17337554
Relationship between global structural parameters and Enzyme Commission hierarchy: implications for function prediction.

PubMed

Boareto, Marcelo; Yamagishi, Michel E B; Caticha, Nestor; Leite, Vitor B P

2012-10-01

In protein databases there is a substantial number of proteins structurally determined but without function annotation. Understanding the relationship between function and structure can be useful to predict function on a large scale. We have analyzed the similarities in global physicochemical parameters for a set of enzymes which were classified according to the four Enzyme Commission (EC) hierarchical levels. Using relevance theory we introduced a distance between proteins in the space of physicochemical characteristics. This was done by minimizing a cost function of the metric tensor built to reflect the EC classification system. Using an unsupervised clustering method on a set of 1025 enzymes, we obtained no relevant clustering formation compatible with EC classification. The distance distributions between enzymes from the same EC group and from different EC groups were compared by histograms. Such analysis was also performed using sequence alignment similarity as a distance. Our results suggest that global structure parameters are not sufficient to segregate enzymes according to EC hierarchy. This indicates that features essential for function are rather local than global. Consequently, methods for predicting function based on global attributes should not obtain high accuracy in main EC classes prediction without relying on similarities between enzymes from training and validation datasets. Furthermore, these results are consistent with a substantial number of studies suggesting that function evolves fundamentally by recruitment, i.e., a same protein motif or fold can be used to perform different enzymatic functions and a few specific amino acids (AAs) are actually responsible for enzyme activity. These essential amino acids should belong to active sites and an effective method for predicting function should be able to recognize them. Copyright © 2012 Elsevier Ltd. All rights reserved.
Carbonic anhydrase inhibition of Schiff base derivative of imino-methyl-naphthalen-2-ol: Synthesis, structure elucidation, molecular docking, dynamic simulation and density functional theory calculations

NASA Astrophysics Data System (ADS)

Abbas, Saghir; Nasir, Hafiza Huma; Zaib, Sumera; Ali, Saqib; Mahmood, Tariq; Ayub, Khurshid; Tahir, Muhammad Nawaz; Iqbal, Jamshed

2018-03-01

In the present study, we have designed and synthesized a Schiff base derivative 3 and characterized by FT-IR, 1H and 13C NMR spectroscopy. Single crystal X-ray diffraction and NMR studies were also performed. The synthetic compound was screened for its inhibitory potential against carbonic anhydrase II. The experimental results were validated by molecular docking and dynamic simulations of compound 3 in the active pocket of enzyme. Important binding interactions with the key residues in the active site of the carbonic anhydrase enzyme were revealed. Moreover, supramolecular assembly of the title compound was analyzed by density functional theory (DFT) calculations. These studies rendered a more clear understanding for the demonstration of novel molecular mechanism involved in CA II inhibition by the synthesized compound.
Crystal genes in a marginal glass-forming system of Ni 50Zr 50

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

Wen, T. Q.; Tang, L.; Sun, Y.

Glass-forming motifs with B2 traits are found. A perfect Ni-centered B33 motif deteriorates the glass-forming ability of Ni 50Zr 50. The marginal glass-forming ability (GFA) of binary Ni-Zr system is an issue to be explained considering the numerous bulk metallic glasses (BMGs) found in the Cu-Zr system. Using molecular dynamics, the structures and dynamics of Ni 50Zr 50 metallic liquid and glass are investigated at the atomistic level. To achieve a well-relaxed glassy sample, sub-T g annealing method is applied and the final sample is closer to the experiments than the models prepared by continuous cooling. With the state-of-the-art structuralmore » analysis tools such as cluster alignment and pair-wise alignment methods, two glass-forming motifs with some mixed traits of the metastable B2 crystalline phase and the crystalline Ni-centered B33 motif are found to be dominant in the undercooled liquid and glass samples. A new chemical order characterization on each short-range order (SRO) structure is accomplished based on the cluster alignment method. The significant amount of the crystalline motif and the few icosahedra in the glassy sample deteriorate the GFA.« less
Crystal genes in a marginal glass-forming system of Ni 50Zr 50

DOE PAGES

Wen, T. Q.; Tang, L.; Sun, Y.; ...

2017-10-17

Glass-forming motifs with B2 traits are found. A perfect Ni-centered B33 motif deteriorates the glass-forming ability of Ni 50Zr 50. The marginal glass-forming ability (GFA) of binary Ni-Zr system is an issue to be explained considering the numerous bulk metallic glasses (BMGs) found in the Cu-Zr system. Using molecular dynamics, the structures and dynamics of Ni 50Zr 50 metallic liquid and glass are investigated at the atomistic level. To achieve a well-relaxed glassy sample, sub-T g annealing method is applied and the final sample is closer to the experiments than the models prepared by continuous cooling. With the state-of-the-art structuralmore » analysis tools such as cluster alignment and pair-wise alignment methods, two glass-forming motifs with some mixed traits of the metastable B2 crystalline phase and the crystalline Ni-centered B33 motif are found to be dominant in the undercooled liquid and glass samples. A new chemical order characterization on each short-range order (SRO) structure is accomplished based on the cluster alignment method. The significant amount of the crystalline motif and the few icosahedra in the glassy sample deteriorate the GFA.« less

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

PubMed

Dzyadevych, Sergei V; Soldatkin, Alexey P; Korpan, Yaroslav I; Arkhypova, Valentyna N; El'skaya, Anna V; Chovelon, Jean-Marc; Martelet, Claude; Jaffrezic-Renault, Nicole

2003-10-01

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.
Stochastic molecular model of enzymatic hydrolysis of cellulose for ethanol production

PubMed Central

2013-01-01

Background During cellulosic ethanol production, cellulose hydrolysis is achieved by synergistic action of cellulase enzyme complex consisting of multiple enzymes with different mode of actions. Enzymatic hydrolysis of cellulose is one of the bottlenecks in the commercialization of the process due to low hydrolysis rates and high cost of enzymes. A robust hydrolysis model that can predict hydrolysis profile under various scenarios can act as an important forecasting tool to improve the hydrolysis process. However, multiple factors affecting hydrolysis: cellulose structure and complex enzyme-substrate interactions during hydrolysis make it diffucult to develop mathematical kinetic models that can simulate hydrolysis in presence of multiple enzymes with high fidelity. In this study, a comprehensive hydrolysis model based on stochastic molecular modeling approch in which each hydrolysis event is translated into a discrete event is presented. The model captures the structural features of cellulose, enzyme properties (mode of actions, synergism, inhibition), and most importantly dynamic morphological changes in the substrate that directly affect the enzyme-substrate interactions during hydrolysis. Results Cellulose was modeled as a group of microfibrils consisting of elementary fibrils bundles, where each elementary fibril was represented as a three dimensional matrix of glucose molecules. Hydrolysis of cellulose was simulated based on Monte Carlo simulation technique. Cellulose hydrolysis results predicted by model simulations agree well with the experimental data from literature. Coefficients of determination for model predictions and experimental values were in the range of 0.75 to 0.96 for Avicel hydrolysis by CBH I action. Model was able to simulate the synergistic action of multiple enzymes during hydrolysis. The model simulations captured the important experimental observations: effect of structural properties, enzyme inhibition and enzyme loadings on the hydrolysis and degree of synergism among enzymes. Conclusions The model was effective in capturing the dynamic behavior of cellulose hydrolysis during action of individual as well as multiple cellulases. Simulations were in qualitative and quantitative agreement with experimental data. Several experimentally observed phenomena were simulated without the need for any additional assumptions or parameter changes and confirmed the validity of using the stochastic molecular modeling approach to quantitatively and qualitatively describe the cellulose hydrolysis. PMID:23638989
Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

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

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less
Time Alignment as a Necessary Step in the Analysis of Sleep Probabilistic Curves

NASA Astrophysics Data System (ADS)

Rošt'áková, Zuzana; Rosipal, Roman

2018-02-01

Sleep can be characterised as a dynamic process that has a finite set of sleep stages during the night. The standard Rechtschaffen and Kales sleep model produces discrete representation of sleep and does not take into account its dynamic structure. In contrast, the continuous sleep representation provided by the probabilistic sleep model accounts for the dynamics of the sleep process. However, analysis of the sleep probabilistic curves is problematic when time misalignment is present. In this study, we highlight the necessity of curve synchronisation before further analysis. Original and in time aligned sleep probabilistic curves were transformed into a finite dimensional vector space, and their ability to predict subjects' age or daily measures is evaluated. We conclude that curve alignment significantly improves the prediction of the daily measures, especially in the case of the S2-related sleep states or slow wave sleep.
Modified compensation algorithm of lever-arm effect and flexural deformation for polar shipborne transfer alignment based on improved adaptive Kalman filter

NASA Astrophysics Data System (ADS)

Wang, Tongda; Cheng, Jianhua; Guan, Dongxue; Kang, Yingyao; Zhang, Wei

2017-09-01

Due to the lever-arm effect and flexural deformation in the practical application of transfer alignment (TA), the TA performance is decreased. The existing polar TA algorithm only compensates a fixed lever-arm without considering the dynamic lever-arm caused by flexural deformation; traditional non-polar TA algorithms also have some limitations. Thus, the performance of existing compensation algorithms is unsatisfactory. In this paper, a modified compensation algorithm of the lever-arm effect and flexural deformation is proposed to promote the accuracy and speed of the polar TA. On the basis of a dynamic lever-arm model and a noise compensation method for flexural deformation, polar TA equations are derived in grid frames. Based on the velocity-plus-attitude matching method, the filter models of polar TA are designed. An adaptive Kalman filter (AKF) is improved to promote the robustness and accuracy of the system, and then applied to the estimation of the misalignment angles. Simulation and experiment results have demonstrated that the modified compensation algorithm based on the improved AKF for polar TA can effectively compensate the lever-arm effect and flexural deformation, and then improve the accuracy and speed of TA in the polar region.
Measurement of Enzyme Isotope Effects.

PubMed

Kholodar, Svetlana A; Ghosh, Ananda K; Kohen, Amnon

2017-01-01

Enzyme isotope effects, or the kinetic effects of "heavy" enzymes, refer to the effect of isotopically labeled protein residues on the enzyme's activity or physical properties. These effects are increasingly employed in the examination of the possible contributions of protein dynamics to enzyme catalysis. One hypothesis assumed that isotopic substitution of all 12 C, 14 N, and nonexchangeable 1 H by 13 C, 15 N, and 2 H, would slow down protein picosecond to femtosecond dynamics without any effect on the system's electrostatics following the Born-Oppenheimer approximation. It was suggested that reduced reaction rates reported for several "heavy" enzymes accords with that hypothesis. However, numerous deviations from the predictions of that hypothesis were also reported. Current studies also attempt to test the role of individual residues by site-specific labeling or by labeling a pattern of residues on activity. It appears that in several systems the protein's fast dynamics are indeed reduced in "heavy" enzymes in a way that reduces the probability of barrier crossing of its chemical step. Other observations, however, indicated that slower protein dynamics are electrostatically altered in isotopically labeled enzymes. Interestingly, these effects appear to be system dependent, thus it might be premature to suggest a general role of "heavy" enzymes' effect on catalysis. © 2017 Elsevier Inc. All rights reserved.
High-speed multiple sequence alignment on a reconfigurable platform.

PubMed

Oliver, Tim; Schmidt, Bertil; Maskell, Douglas; Nathan, Darran; Clemens, Ralf

2006-01-01

Progressive alignment is a widely used approach to compute multiple sequence alignments (MSAs). However, aligning several hundred sequences by popular progressive alignment tools requires hours on sequential computers. Due to the rapid growth of sequence databases biologists have to compute MSAs in a far shorter time. In this paper we present a new approach to MSA on reconfigurable hardware platforms to gain high performance at low cost. We have constructed a linear systolic array to perform pairwise sequence distance computations using dynamic programming. This results in an implementation with significant runtime savings on a standard FPGA.
Model for the orientational ordering of the plant microtubule cortical array

NASA Astrophysics Data System (ADS)

Hawkins, Rhoda J.; Tindemans, Simon H.; Mulder, Bela M.

2010-07-01

The plant microtubule cortical array is a striking feature of all growing plant cells. It consists of a more or less homogeneously distributed array of highly aligned microtubules connected to the inner side of the plasma membrane and oriented transversely to the cell growth axis. Here, we formulate a continuum model to describe the origin of orientational order in such confined arrays of dynamical microtubules. The model is based on recent experimental observations that show that a growing cortical microtubule can interact through angle dependent collisions with pre-existing microtubules that can lead either to co-alignment of the growth, retraction through catastrophe induction or crossing over the encountered microtubule. We identify a single control parameter, which is fully determined by the nucleation rate and intrinsic dynamics of individual microtubules. We solve the model analytically in the stationary isotropic phase, discuss the limits of stability of this isotropic phase, and explicitly solve for the ordered stationary states in a simplified version of the model.
All Inkjet-Printed Amperometric Multiplexed Biosensors Based on Nanostructured Conductive Hydrogel Electrodes.

PubMed

Li, Lanlan; Pan, Lijia; Ma, Zhong; Yan, Ke; Cheng, Wen; Shi, Yi; Yu, Guihua

2018-06-13

Multiplexing, one of the main trends in biosensors, aims to detect several analytes simultaneously by integrating miniature sensors on a chip. However, precisely depositing electrode materials and selective enzymes on distinct microelectrode arrays remains an obstacle to massively produced multiplexed sensors. Here, we report on a "drop-on-demand" inkjet printing process to fabricate multiplexed biosensors based on nanostructured conductive hydrogels in which the electrode material and several kinds of enzymes were printed on the electrode arrays one by one by employing a multinozzle inkjet system. The whole inkjet printing process can be finished within three rounds of printing and only one round of alignment. For a page of sensor arrays containing 96 working electrodes, the printing process took merely ∼5 min. The multiplexed assays can detect glucose, lactate, and triglycerides in real time with good selectivity and high sensitivity, and the results in phosphate buffer solutions and calibration serum samples are comparable. The inkjet printing process exhibited advantages of high efficiency and accuracy, which opens substantial possibilities for massive fabrication of integrated multiplexed biosensors for human health monitoring.
Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.
Potent Inhibitors of the Hepatitis C Virus NS3 Protease: Design and Synthesis of Macrocyclic Substrate-Based [beta]-Strand Mimics

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

Goudreau, Nathalie; Brochu, Christian; Cameron, Dale R.

2008-06-30

The virally encoded NS3 protease is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. The design and synthesis of 15-membered ring {beta}-strand mimics which are capable of inhibiting the interactions between the HCV NS3 protease enzyme and its polyprotein substrate will be described. The binding interactions between a macrocyclic ligand and the enzyme were explored by NMR and molecular dynamics, and a model of the ligand/enzyme complex was developed.
Using Information Communication Technologies to Develop Dynamic Curriculum Frameworks for Diverse Cohorts: A Case Study from Event Management

ERIC Educational Resources Information Center

Hadley, Bree Jamila

2012-01-01

This article investigates the role of information communication technologies (ICTs) in establishing a well-aligned, authentic learning environment for a diverse cohort of non-cognate and cognate students studying event management in a higher education context. Based on a case study which examined the way ICTs assisted in accommodating diverse…
Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

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

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth

2009-09-11

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft aremore » more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less
Quantitative phase microscopy for cellular dynamics based on transport of intensity equation.

PubMed

Li, Ying; Di, Jianglei; Ma, Chaojie; Zhang, Jiwei; Zhong, Jinzhan; Wang, Kaiqiang; Xi, Teli; Zhao, Jianlin

2018-01-08

We demonstrate a simple method for quantitative phase imaging of tiny transparent objects such as living cells based on the transport of intensity equation. The experiments are performed using an inverted bright field microscope upgraded with a flipping imaging module, which enables to simultaneously create two laterally separated images with unequal defocus distances. This add-on module does not include any lenses or gratings and is cost-effective and easy-to-alignment. The validity of this method is confirmed by the measurement of microlens array and human osteoblastic cells in culture, indicating its potential in the applications of dynamically measuring living cells and other transparent specimens in a quantitative, non-invasive and label-free manner.
Static and dynamic half-life and lifetime molecular turnover of enzymes.

PubMed

Miyawaki, Osato; Kanazawa, Tsukasa; Maruyama, Chika; Dozen, Michiko

2017-01-01

The static half-life of an enzyme is the half-life of a free enzyme not working without substrate and the dynamic half-life is that of an active enzyme working with plenty amount of substrate. These two half-lives were measured and compared for glucoamylase (GA) and β-galactosidase (BG). The dynamic half-life was much longer than the static half-life by one to three orders of magnitude for both enzymes. For BG, the half-life of the enzyme physically entrapped in a membrane reactor was also measured. In this case also, the half-life of BG in the membrane reactor was much longer than the free enzyme without substrate. These results suggest the large difference in stabilities between the free enzyme and the enzyme-substrate complex. This may be related to the natural enzyme metabolism. According to the difference in half-life, the lifetime molecular turnover (LMT), which is the number of product molecules produced by a single molecule of enzyme until it loses its activity completely, was much higher by one to four orders of magnitude for the active enzyme than the free enzyme. The concept of LMT, proposed here, will be important in bioreactor operations with or without immobilization. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Effects of Matrix Alignment and Mechanical Constraints on Cellular Behavior in 3D Engineered Microtissues

NASA Astrophysics Data System (ADS)

Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

The adhesion of cells to the extracellular matrix (ECM) plays a crucial role in a variety of cellular functions. The main building blocks of the ECM are 3D networks of fibrous proteins whose structure and alignments varies with tissue type. However, the impact of ECM alignment on cellular behaviors such as cell adhesion, spreading, extension and mechanics remains poorly understood. We present results on the development of a microtissue-based system that enables control of the structure, orientation, and degree of fibrillar alignment in 3D fibroblast-populated collagen gels. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of elastic pillars. The contractile action of the cells leads to controlled alignment of the fibrous collagen, depending on the number and location of the pillars in each well. The pillars are elastic, and are utilized to measure the contractile forces of the microtissues, and by incorporating magnetic material in selected pillars, time-varying forces can be applied to the tissues for dynamic stimulation and measurement of mechanical properties. Results on the effects of varying pillar shape, spacing, location, and stiffness on microtissue organization and contractility will be presented. This work is supported by NSF CMMI-1463011.
Tetrahedron deformation and alignment of perceived vorticity and strain in a turbulent flow

NASA Astrophysics Data System (ADS)

Pumir, Alain; Bodenschatz, Eberhard; Xu, Haitao

2013-03-01

We describe the structure and dynamics of turbulence by the scale-dependent perceived velocity gradient tensor as supported by following four tracers, i.e., fluid particles, that initially form a regular tetrahedron. We report results from experiments in a von Kármán swirling water flow and from numerical simulations of the incompressible Navier-Stokes equation. We analyze the statistics and the dynamics of the perceived rate of strain tensor and vorticity for initially regular tetrahedron of size r0 from the dissipative to the integral scale. Just as for the true velocity gradient, at any instant, the perceived vorticity is also preferentially aligned with the intermediate eigenvector of the perceived rate of strain. However, in the perceived rate of strain eigenframe fixed at a given time t = 0, the perceived vorticity evolves in time such as to align with the strongest eigendirection at t = 0. This also applies to the true velocity gradient. The experimental data at the higher Reynolds number suggests the existence of a self-similar regime in the inertial range. In particular, the dynamics of alignment of the perceived vorticity and strain can be rescaled by t0, the turbulence time scale of the flow when the scale r0 is in the inertial range. For smaller Reynolds numbers we found the dynamics to be scale dependent.
Terminator field-aligned current system: A new finding from model-assimilated data set (MADS)

NASA Astrophysics Data System (ADS)

Zhu, L.; Schunk, R. W.; Scherliess, L.; Sojka, J. J.; Gardner, L. C.; Eccles, J. V.; Rice, D.

2013-12-01

Physics-based data assimilation models have been recognized by the space science community as the most accurate approach to specify and forecast the space weather of the solar-terrestrial environment. The model-assimilated data sets (MADS) produced by these models constitute an internally consistent time series of global three-dimensional fields whose accuracy can be estimated. Because of its internal consistency of physics and completeness of descriptions on the status of global systems, the MADS has also been a powerful tool to identify the systematic errors in measurements, reveal the missing physics in physical models, and discover the important dynamical physical processes that are inadequately observed or missed by measurements due to observational limitations. In the past years, we developed a data assimilation model for the high-latitude ionospheric plasma dynamics and electrodynamics. With a set of physical models, an ensemble Kalman filter, and the ingestion of data from multiple observations, the data assimilation model can produce a self-consistent time-series of the complete descriptions of the global high-latitude ionosphere, which includes the convection electric field, horizontal and field-aligned currents, conductivity, as well as 3-D plasma densities and temperatures, In this presentation, we will show a new field-aligned current system discovered from the analysis of the MADS produced by our data assimilation model. This new current system appears and develops near the ionospheric terminator. The dynamical features of this current system will be described and its connection to the active role of the ionosphere in the M-I coupling will be discussed.
Multi-channel dynamics in high harmonic generation of aligned CO2: ab initio analysis with time-dependent B-spline algebraic diagrammatic construction.

PubMed

Ruberti, M; Decleva, P; Averbukh, V

2018-03-28

Here we present a fully ab initio study of the high-order harmonic generation (HHG) spectrum of aligned CO 2 molecules. The calculations have been performed by using the molecular time-dependent (TD) B-spline algebraic diagrammatic construction (ADC) method. We quantitatively study how the sub-cycle laser-driven multi-channel dynamics, as reflected in the position of the dynamical minimum in the HHG spectrum, is affected by the full inclusion of both correlation-driven and laser-driven dipole interchannel couplings. We calculate channel-resolved spectral intensities as well as the phase differences between contributions of the different ionization-recombination channels to the total HHG spectrum. Our results show that electron correlation effectively controls the relative contributions of the different channels to the total HHG spectrum, leading to the opening of the new ones (1 2 Π u , 1 2 Σ), previously disregarded for the aligned molecular setup. We conclude that inclusion of many-electron effects into the theoretical interpretation of molecular HHG spectra is essential in order to correctly extract ultrafast electron dynamics using HHG spectroscopy.
Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis

PubMed Central

Zaccardi, Margot J; O'Rourke, Kathleen F; Yezdimer, Eric M; Loggia, Laura J; Woldt, Svenja; Boehr, David D

2014-01-01

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site β1α1 and β2α2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements. PMID:24403092

Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization.

PubMed

Hinsberger, Stefan; Hüsecken, Kristina; Groh, Matthias; Negri, Matthias; Haupenthal, Jörg; Hartmann, Rolf W

2013-11-14

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.
Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

PubMed Central

2016-01-01

SUMMARY Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism. PMID:27074917
Dynamic optimization of metabolic networks coupled with gene expression.

PubMed

Waldherr, Steffen; Oyarzún, Diego A; Bockmayr, Alexander

2015-01-21

The regulation of metabolic activity by tuning enzyme expression levels is crucial to sustain cellular growth in changing environments. Metabolic networks are often studied at steady state using constraint-based models and optimization techniques. However, metabolic adaptations driven by changes in gene expression cannot be analyzed by steady state models, as these do not account for temporal changes in biomass composition. Here we present a dynamic optimization framework that integrates the metabolic network with the dynamics of biomass production and composition. An approximation by a timescale separation leads to a coupled model of quasi-steady state constraints on the metabolic reactions, and differential equations for the substrate concentrations and biomass composition. We propose a dynamic optimization approach to determine reaction fluxes for this model, explicitly taking into account enzyme production costs and enzymatic capacity. In contrast to the established dynamic flux balance analysis, our approach allows predicting dynamic changes in both the metabolic fluxes and the biomass composition during metabolic adaptations. Discretization of the optimization problems leads to a linear program that can be efficiently solved. We applied our algorithm in two case studies: a minimal nutrient uptake network, and an abstraction of core metabolic processes in bacteria. In the minimal model, we show that the optimized uptake rates reproduce the empirical Monod growth for bacterial cultures. For the network of core metabolic processes, the dynamic optimization algorithm predicted commonly observed metabolic adaptations, such as a diauxic switch with a preference ranking for different nutrients, re-utilization of waste products after depletion of the original substrate, and metabolic adaptation to an impending nutrient depletion. These examples illustrate how dynamic adaptations of enzyme expression can be predicted solely from an optimization principle. Copyright © 2014 Elsevier Ltd. All rights reserved.
Functional Roles of Slow Enzyme Conformational Changes in Network Dynamics

PubMed Central

Wu, Zhanghan; Xing, Jianhua

2012-01-01

Extensive studies from different fields reveal that many macromolecules, especially enzymes, show slow transitions among different conformations. This phenomenon is named such things as dynamic disorder, heterogeneity, hysteretic or mnemonic enzymes across these different fields, and has been directly demonstrated by single molecule enzymology and NMR studies recently. We analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can filter upstream network noises, and can either resonantly respond to the system stimulus at certain frequencies or respond adaptively for sustained input signals of the network fluctuations. It thus can serve as a basic functional motif with properties that are normally for larger intermolecular networks in the field of systems biology. We further analyzed examples including enzymes functioning against pH fluctuations, metabolic state change of Artemia embryos, and kinetic insulation of fluctuations in metabolic networks. The study also suggests that hysteretic enzymes may be building blocks of synthetic networks with various properties such as narrow-banded filtering. The work fills the missing gap between studies on enzyme biophysics and network level dynamics, and reveals that the coupling between the two is functionally important; it also suggests that the conformational dynamics of some enzymes may be evolutionally selected. PMID:23009855
Alignment-free Transcriptomic and Metatranscriptomic Comparison Using Sequencing Signatures with Variable Length Markov Chains.

PubMed

Liao, Weinan; Ren, Jie; Wang, Kun; Wang, Shun; Zeng, Feng; Wang, Ying; Sun, Fengzhu

2016-11-23

The comparison between microbial sequencing data is critical to understand the dynamics of microbial communities. The alignment-based tools analyzing metagenomic datasets require reference sequences and read alignments. The available alignment-free dissimilarity approaches model the background sequences with Fixed Order Markov Chain (FOMC) yielding promising results for the comparison of microbial communities. However, in FOMC, the number of parameters grows exponentially with the increase of the order of Markov Chain (MC). Under a fixed high order of MC, the parameters might not be accurately estimated owing to the limitation of sequencing depth. In our study, we investigate an alternative to FOMC to model background sequences with the data-driven Variable Length Markov Chain (VLMC) in metatranscriptomic data. The VLMC originally designed for long sequences was extended to apply to high-throughput sequencing reads and the strategies to estimate the corresponding parameters were developed. The flexible number of parameters in VLMC avoids estimating the vast number of parameters of high-order MC under limited sequencing depth. Different from the manual selection in FOMC, VLMC determines the MC order adaptively. Several beta diversity measures based on VLMC were applied to compare the bacterial RNA-Seq and metatranscriptomic datasets. Experiments show that VLMC outperforms FOMC to model the background sequences in transcriptomic and metatranscriptomic samples. A software pipeline is available at https://d2vlmc.codeplex.com.
Molecular modeling of cytochrome P450 3A4

NASA Astrophysics Data System (ADS)

Szklarz, Grazyna D.; Halpert, James R.

1997-05-01

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6β-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.
Dynamic Scattering Mode LCDs

NASA Astrophysics Data System (ADS)

Bahadur, Birendra

The following sections are included: * INTRODUCTION * CELL DESIGNING * EXPERIMENTAL OBSERVATIONS IN NEMATICS RELATED WITH DYNAMIC SCATTERING * Experimental Observations at D.C. Field and Electrode Effects * Experimental Observation at Low Frequency A.C. Fields * Homogeneously Aligned Nematic Regime * Williams Domains * Dynamic Scattering * Experimental Observation at High Frequency A.C. Field * Other Experimental Observations * THEORETICAL INTERPRETATIONS * Felici Model * Carr-Helfrich Model * D.C. Excitation * Dubois-Violette, de Gennes and Parodi Model * Low Freqency or Conductive Regime * High Frequency or Dielectric Regime * DYNAMIC SCATTERING IN SMECRIC A PHASE * ELECTRO-OPTICAL CHARACTERISTICS AND LIMITATIONS * Contrast Ratio vs. Voltage, Viewing Angle, Cell Gap, Wavelength and Temperature * Display Current vs. Voltage, Cell Gap and Temperature * Switching Time * Effect of Alignment * Effect of Conductivity, Temperature and Frequency * Addressing of DSM LCDs * Limitations of DSM LCDs * ACKNOWLEDGEMENTS * REFERENCES
DOE Office of Scientific and Technical Information (OSTI.GOV)

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less
Paramagnetic alignment of small grains: A novel method for measuring interstellar magnetic fields

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

Hoang, Thiem; Martin, P. G.; Lazarian, A.

2014-07-20

We present a novel method to measure the strength of interstellar magnetic fields using ultraviolet (UV) polarization of starlight that is in part produced by weakly aligned, small dust grains. We begin with calculating the degrees of the paramagnetic alignment of small (size a ∼ 0.01 μm) and very small (a ∼ 0.001 μm) grains in the interstellar magnetic field due to the Davis-Greenstein relaxation and resonance relaxation. To calculate the degrees of paramagnetic alignment, we use Langevin equations and take into account various interaction processes essential for the rotational dynamics of small grains. We find that the alignment ofmore » small grains is necessary to reproduce the observed polarization in the UV, although the polarization arising from these small grains is negligible at the optical and infrared (IR) wavelengths. Based on fitting theoretical models to observed extinction and polarization curves, we find that the best-fit model for the case with the peak wavelength of polarization λ{sub max} < 0.55 μm requires a higher degree of alignment of small grains than for the typical case with λ{sub max} = 0.55 μm. We interpret the correlation between the systematic increase of the UV polarization relative to maximum polarization (i.e., of p(6 μm{sup –1})/p{sub max}) with λ{sub max}{sup −1} for cases of low λ{sub max} by appealing to the higher degree of alignment of small grains. We utilize the correlation of the paramagnetic alignment of small grains with the magnetic field strength B to suggest a new way to measure B using the observable parameters λ{sub max} and p(6 μm{sup –1})/p{sub max}.« less
Design Principles of DNA Enzyme-Based Walkers: Translocation Kinetics and Photoregulation.

PubMed

Cha, Tae-Gon; Pan, Jing; Chen, Haorong; Robinson, Heather N; Li, Xiang; Mao, Chengde; Choi, Jong Hyun

2015-07-29

Dynamic DNA enzyme-based walkers complete their stepwise movements along the prescribed track through a series of reactions, including hybridization, enzymatic cleavage, and strand displacement; however, their overall translocation kinetics is not well understood. Here, we perform mechanistic studies to elucidate several key parameters that govern the kinetics and processivity of DNA enzyme-based walkers. These parameters include DNA enzyme core type and structure, upper and lower recognition arm lengths, and divalent metal cation species and concentration. A theoretical model is developed within the framework of single-molecule kinetics to describe overall translocation kinetics as well as each reaction step. A better understanding of kinetics and design parameters enables us to demonstrate a walker movement near 5 μm at an average speed of ∼1 nm s(-1). We also show that the translocation kinetics of DNA walkers can be effectively controlled by external light stimuli using photoisomerizable azobenzene moieties. A 2-fold increase in the cleavage reaction is observed when the hairpin stems of enzyme catalytic cores are open under UV irradiation. This study provides general design guidelines to construct highly processive, autonomous DNA walker systems and to regulate their translocation kinetics, which would facilitate the development of functional DNA walkers.
Development and alignment of undergraduate medical curricula in a web-based, dynamic Learning Opportunities, Objectives and Outcome Platform (LOOOP).

PubMed

Balzer, Felix; Hautz, Wolf E; Spies, Claudia; Bietenbeck, Andreas; Dittmar, Martin; Sugiharto, Firman; Lehmann, Lars; Eisenmann, Dorothea; Bubser, Florian; Stieg, Markus; Hanfler, Sven; Georg, Waltraud; Tekian, Ara; Ahlers, Olaf

2016-01-01

This study presents a web-based method and its interface ensuring alignment of all parts of a curriculum map including competencies, objectives, teaching and assessment methods, workload and patient availability. Needs, acceptance and effectiveness are shown through a nine-year study. After a comprehensive needs assessment, the curriculum map and a web-based interface "Learning Opportunities, Objectives and Outcome Platform" (LOOOP) were developed according to Harden's conceptual framework of 10-steps for curriculum mapping. The outcome was measured by surveys and results of interdisciplinary MCQ-assessments. The usage rates and functionalities were analysed. The implementation of LOOOP was significantly associated with improved perception of the curriculum structure by teachers and students, quality of defined objectives and their alignment with teaching and assessment, usage by students to prepare examinations and their scores in interdisciplinary MCQ-assessment. Additionally, LOOOP improved the curriculum coordination by faculty, and assisted departments for identifying patient availability for clinical training. LOOOP is well accepted among students and teachers, has positive effect on curriculum development, facilitates effective utilisation of educational resources and improves student's outcomes. Currently, LOOOP is used in five undergraduate medical curricula including 85,000 mapped learning opportunities (lectures, seminars), 5000 registered users (students, teachers) and 380,000 yearly page-visits.
Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

PubMed

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics and function with high specificity, offering substantial advantages in both sensitivity and specificity over conventional detection methods. The screening of nuclease, methyltransferase, protease, and kinase activities can be colorimetrically performed in a straightforward manner. Finally, we discuss examples of colorimetric assays for metal ions and small molecules that constitute important advances toward visual monitoring of enzyme catalytic functions and gene expression. Although these enzyme-assisted assay methods hold great promise for myriad applications in biomedicine and bioimaging, the application of the described techniques in vivo faces formidable challenges. In addition, researchers do not fully understand the interactions of gold nanoparticles with enzyme molecules. This understanding will require the development of new techniques to probe enzyme substrate dynamics at the particle interface with higher spatial resolution and chemical specificity.
Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.
Railway Tunnel Clearance Inspection Method Based on 3D Point Cloud from Mobile Laser Scanning

PubMed Central

Zhou, Yuhui; Wang, Shaohua; Mei, Xi; Yin, Wangling; Lin, Chunfeng; Mao, Qingzhou

2017-01-01

Railway tunnel clearance is directly related to the safe operation of trains and upgrading of freight capacity. As more and more railway are put into operation and the operation is continuously becoming faster, the railway tunnel clearance inspection should be more precise and efficient. In view of the problems existing in traditional tunnel clearance inspection methods, such as low density, slow speed and a lot of manual operations, this paper proposes a tunnel clearance inspection approach based on 3D point clouds obtained by a mobile laser scanning system (MLS). First, a dynamic coordinate system for railway tunnel clearance inspection has been proposed. A rail line extraction algorithm based on 3D linear fitting is implemented from the segmented point cloud to establish a dynamic clearance coordinate system. Second, a method to seamlessly connect all rail segments based on the railway clearance restrictions, and a seamless rail alignment is formed sequentially from the middle tunnel section to both ends. Finally, based on the rail alignment and the track clearance coordinate system, different types of clearance frames are introduced for intrusion operation with the tunnel section to realize the tunnel clearance inspection. By taking the Shuanghekou Tunnel of the Chengdu–Kunming Railway as an example, when the clearance inspection is carried out by the method mentioned herein, its precision can reach 0.03 m, and difference types of clearances can be effectively calculated. This method has a wide application prospects. PMID:28880232
Primary structure of inorganic polyphosphate/ATP-NAD kinase from Micrococcus flavus, and occurrence of substrate inorganic polyphosphate for the enzyme.

PubMed

Kawai, Shigeyuki; Mori, Shigetarou; Murata, Kousaku

2003-08-01

The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.
Combining peak- and chromatogram-based retention time alignment algorithms for multiple chromatography-mass spectrometry datasets.

PubMed

Hoffmann, Nils; Keck, Matthias; Neuweger, Heiko; Wilhelm, Mathias; Högy, Petra; Niehaus, Karsten; Stoye, Jens

2012-08-27

Modern analytical methods in biology and chemistry use separation techniques coupled to sensitive detectors, such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). These hyphenated methods provide high-dimensional data. Comparing such data manually to find corresponding signals is a laborious task, as each experiment usually consists of thousands of individual scans, each containing hundreds or even thousands of distinct signals. In order to allow for successful identification of metabolites or proteins within such data, especially in the context of metabolomics and proteomics, an accurate alignment and matching of corresponding features between two or more experiments is required. Such a matching algorithm should capture fluctuations in the chromatographic system which lead to non-linear distortions on the time axis, as well as systematic changes in recorded intensities. Many different algorithms for the retention time alignment of GC-MS and LC-MS data have been proposed and published, but all of them focus either on aligning previously extracted peak features or on aligning and comparing the complete raw data containing all available features. In this paper we introduce two algorithms for retention time alignment of multiple GC-MS datasets: multiple alignment by bidirectional best hits peak assignment and cluster extension (BIPACE) and center-star multiple alignment by pairwise partitioned dynamic time warping (CeMAPP-DTW). We show how the similarity-based peak group matching method BIPACE may be used for multiple alignment calculation individually and how it can be used as a preprocessing step for the pairwise alignments performed by CeMAPP-DTW. We evaluate the algorithms individually and in combination on a previously published small GC-MS dataset studying the Leishmania parasite and on a larger GC-MS dataset studying grains of wheat (Triticum aestivum). We have shown that BIPACE achieves very high precision and recall and a very low number of false positive peak assignments on both evaluation datasets. CeMAPP-DTW finds a high number of true positives when executed on its own, but achieves even better results when BIPACE is used to constrain its search space. The source code of both algorithms is included in the OpenSource software framework Maltcms, which is available from http://maltcms.sf.net. The evaluation scripts of the present study are available from the same source.
Combining peak- and chromatogram-based retention time alignment algorithms for multiple chromatography-mass spectrometry datasets

PubMed Central

2012-01-01

Background Modern analytical methods in biology and chemistry use separation techniques coupled to sensitive detectors, such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). These hyphenated methods provide high-dimensional data. Comparing such data manually to find corresponding signals is a laborious task, as each experiment usually consists of thousands of individual scans, each containing hundreds or even thousands of distinct signals. In order to allow for successful identification of metabolites or proteins within such data, especially in the context of metabolomics and proteomics, an accurate alignment and matching of corresponding features between two or more experiments is required. Such a matching algorithm should capture fluctuations in the chromatographic system which lead to non-linear distortions on the time axis, as well as systematic changes in recorded intensities. Many different algorithms for the retention time alignment of GC-MS and LC-MS data have been proposed and published, but all of them focus either on aligning previously extracted peak features or on aligning and comparing the complete raw data containing all available features. Results In this paper we introduce two algorithms for retention time alignment of multiple GC-MS datasets: multiple alignment by bidirectional best hits peak assignment and cluster extension (BIPACE) and center-star multiple alignment by pairwise partitioned dynamic time warping (CeMAPP-DTW). We show how the similarity-based peak group matching method BIPACE may be used for multiple alignment calculation individually and how it can be used as a preprocessing step for the pairwise alignments performed by CeMAPP-DTW. We evaluate the algorithms individually and in combination on a previously published small GC-MS dataset studying the Leishmania parasite and on a larger GC-MS dataset studying grains of wheat (Triticum aestivum). Conclusions We have shown that BIPACE achieves very high precision and recall and a very low number of false positive peak assignments on both evaluation datasets. CeMAPP-DTW finds a high number of true positives when executed on its own, but achieves even better results when BIPACE is used to constrain its search space. The source code of both algorithms is included in the OpenSource software framework Maltcms, which is available from http://maltcms.sf.net. The evaluation scripts of the present study are available from the same source. PMID:22920415
Interactive software tool to comprehend the calculation of optimal sequence alignments with dynamic programming.

PubMed

Ibarra, Ignacio L; Melo, Francisco

2010-07-01

Dynamic programming (DP) is a general optimization strategy that is successfully used across various disciplines of science. In bioinformatics, it is widely applied in calculating the optimal alignment between pairs of protein or DNA sequences. These alignments form the basis of new, verifiable biological hypothesis. Despite its importance, there are no interactive tools available for training and education on understanding the DP algorithm. Here, we introduce an interactive computer application with a graphical interface, for the purpose of educating students about DP. The program displays the DP scoring matrix and the resulting optimal alignment(s), while allowing the user to modify key parameters such as the values in the similarity matrix, the sequence alignment algorithm version and the gap opening/extension penalties. We hope that this software will be useful to teachers and students of bioinformatics courses, as well as researchers who implement the DP algorithm for diverse applications. The software is freely available at: http:/melolab.org/sat. The software is written in the Java computer language, thus it runs on all major platforms and operating systems including Windows, Mac OS X and LINUX. All inquiries or comments about this software should be directed to Francisco Melo at fmelo@bio.puc.cl.
Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.

PubMed

Kürten, Charlotte; Syrén, Per-Olof

2016-01-16

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.
GateKeeper: a new hardware architecture for accelerating pre-alignment in DNA short read mapping.

PubMed

Alser, Mohammed; Hassan, Hasan; Xin, Hongyi; Ergin, Oguz; Mutlu, Onur; Alkan, Can

2017-11-01

High throughput DNA sequencing (HTS) technologies generate an excessive number of small DNA segments -called short reads- that cause significant computational burden. To analyze the entire genome, each of the billions of short reads must be mapped to a reference genome based on the similarity between a read and 'candidate' locations in that reference genome. The similarity measurement, called alignment, formulated as an approximate string matching problem, is the computational bottleneck because: (i) it is implemented using quadratic-time dynamic programming algorithms and (ii) the majority of candidate locations in the reference genome do not align with a given read due to high dissimilarity. Calculating the alignment of such incorrect candidate locations consumes an overwhelming majority of a modern read mapper's execution time. Therefore, it is crucial to develop a fast and effective filter that can detect incorrect candidate locations and eliminate them before invoking computationally costly alignment algorithms. We propose GateKeeper, a new hardware accelerator that functions as a pre-alignment step that quickly filters out most incorrect candidate locations. GateKeeper is the first design to accelerate pre-alignment using Field-Programmable Gate Arrays (FPGAs), which can perform pre-alignment much faster than software. When implemented on a single FPGA chip, GateKeeper maintains high accuracy (on average >96%) while providing, on average, 90-fold and 130-fold speedup over the state-of-the-art software pre-alignment techniques, Adjacency Filter and Shifted Hamming Distance (SHD), respectively. The addition of GateKeeper as a pre-alignment step can reduce the verification time of the mrFAST mapper by a factor of 10. https://github.com/BilkentCompGen/GateKeeper. mohammedalser@bilkent.edu.tr or onur.mutlu@inf.ethz.ch or calkan@cs.bilkent.edu.tr. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.

PubMed

Kyoung, Minjoung; Russell, Sarah J; Kohnhorst, Casey L; Esemoto, Nopondo N; An, Songon

2015-01-27

Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly.
Taking Ockham's razor to enzyme dynamics and catalysis.

PubMed

Glowacki, David R; Harvey, Jeremy N; Mulholland, Adrian J

2012-01-29

The role of protein dynamics in enzyme catalysis is a matter of intense current debate. Enzyme-catalysed reactions that involve significant quantum tunnelling can give rise to experimental kinetic isotope effects with complex temperature dependences, and it has been suggested that standard statistical rate theories, such as transition-state theory, are inadequate for their explanation. Here we introduce aspects of transition-state theory relevant to the study of enzyme reactivity, taking cues from chemical kinetics and dynamics studies of small molecules in the gas phase and in solution--where breakdowns of statistical theories have received significant attention and their origins are relatively better understood. We discuss recent theoretical approaches to understanding enzyme activity and then show how experimental observations for a number of enzymes may be reproduced using a transition-state-theory framework with physically reasonable parameters. Essential to this simple model is the inclusion of multiple conformations with different reactivity.
Illuminating the Mechanistic Roles of Enzyme Conformational Dynamics

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

Hanson, Jeffrey A.; Dunderstadt, Karl; Watkins, Lucas P.

2007-11-13

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme’s lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK’s domain movements on its catalytic time scale. To quantitatively measure the enzyme’s entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly,more » the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme’s conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme’s rate-limiting step.« less
6DOF Testing of the SLS Inertial Navigation Unit

NASA Technical Reports Server (NTRS)

Geohagan, Kevin; Bernard, Bill; Oliver, T. Emerson; Leggett, Jared; Strickland, Dennis

2018-01-01

The Navigation System on the NASA Space Launch System (SLS) Block 1 vehicle performs initial alignment of the Inertial Navigation System (INS) navigation frame through gyrocompass alignment (GCA). Because the navigation architecture for the SLS Block 1 vehicle is a purely inertial system, the accuracy of the achieved orbit relative to mission requirements is very sensitive to initial alignment accuracy. The assessment of this sensitivity and many others via simulation is a part of the SLS Model-Based Design and Model-Based Requirements approach. As a part of the aforementioned, 6DOF Monte Carlo simulation is used in large part to develop and demonstrate verification of program requirements. To facilitate this and the GN&C flight software design process, an SLS-Program-controlled Design Math Model (DMM) of the SLS INS was developed by the SLS Navigation Team. The SLS INS model implements all of the key functions of the hardware-namely, GCA, inertial navigation, and FDIR (Fault Detection, Isolation, and Recovery)-in support of SLS GN&C design requirements verification. Despite the strong sensitivity to initial alignment, GCA accuracy requirements were not verified by test due to program cost and schedule constraints. Instead, the system relies upon assessments performed using the SLS INS model. In order to verify SLS program requirements by analysis, the SLS INS model is verified and validated against flight hardware. In lieu of direct testing of GCA accuracy in support of requirement verification, the SLS Navigation Team proposed and conducted an engineering test to, among other things, validate the GCA performance and overall behavior of the SLS INS model through comparison with test data. This paper will detail dynamic hardware testing of the SLS INS, conducted by the SLS Navigation Team at Marshall Space Flight Center's 6DOF Table Facility, in support of GCA performance characterization and INS model validation. A 6-DOF motion platform was used to produce 6DOF pad twist and sway dynamics while a simulated SLS flight computer communicated with the INS. Tests conducted include an evaluation of GCA algorithm robustness to increasingly dynamic pad environments, an examination of GCA algorithm stability and accuracy over long durations, and a long-duration static test to gather enough data for Allan Variance analysis. Test setup, execution, and data analysis will be discussed, including analysis performed in support of SLS INS model validation.
The first genome-level transcriptome of the wood-degrading fungus Phanerochaete chrysosporium grown on red oak.

PubMed

Sato, Shin; Feltus, F Alex; Iyer, Prashanti; Tien, Ming

2009-06-01

As part of an effort to determine all the gene products involved in wood degradation, we have performed massively parallel pyrosequencing on an expression library from the white rot fungus Phanerochaete chrysosporium grown in shallow stationary cultures with red oak as the carbon source. Approximately 48,000 high quality sequence tags (246 bp average length) were generated. 53% of the sequence tags aligned to 4,262 P. chrysosporium gene models, and an additional 18.5% of the tags reliably aligned to the P. chrysosporium genome providing evidence for 961 putative novel fragmented gene models. Due to their role in lignocellulose degradation, the secreted proteins were focused upon. Our results show that the four enzymes required for cellulose degradation: endocellulase, exocellulase CBHI, exocellulase CBHII, and beta-glucosidase are all produced. For hemicellulose degradation, not all known enzymes were produced, but endoxylanases, acetyl xylan esterases and mannosidases were detected. For lignin degradation, the role of peroxidases has been questioned; however, our results show that lignin peroxidase is highly expressed along with the H(2)O(2) generating enzyme, alcohol oxidase. The transcriptome snapshot reveals that H(2)O(2) generation and utilization are central in wood degradation. Our results also reveal new transcripts that encode extracellular proteins with no known function.
An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis*

PubMed Central

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

2016-01-01

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. PMID:27810896
An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

DOE PAGES

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; ...

2016-11-03

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less
Rigid-body motion correction of the liver in image reconstruction for golden-angle stack-of-stars DCE MRI.

PubMed

Johansson, Adam; Balter, James; Cao, Yue

2018-03-01

Respiratory motion can affect pharmacokinetic perfusion parameters quantified from liver dynamic contrast-enhanced MRI. Image registration can be used to align dynamic images after reconstruction. However, intra-image motion blur remains after alignment and can alter the shape of contrast-agent uptake curves. We introduce a method to correct for inter- and intra-image motion during image reconstruction. Sixteen liver dynamic contrast-enhanced MRI examinations of nine subjects were performed using a golden-angle stack-of-stars sequence. For each examination, an image time series with high temporal resolution but severe streak artifacts was reconstructed. Images were aligned using region-limited rigid image registration within a region of interest covering the liver. The transformations resulting from alignment were used to correct raw data for motion by modulating and rotating acquired lines in k-space. The corrected data were then reconstructed using view sharing. Portal-venous input functions extracted from motion-corrected images had significantly greater peak signal enhancements (mean increase: 16%, t-test, P < 0.001) than those from images aligned using image registration after reconstruction. In addition, portal-venous perfusion maps estimated from motion-corrected images showed fewer artifacts close to the edge of the liver. Motion-corrected image reconstruction restores uptake curves distorted by motion. Motion correction also reduces motion artifacts in estimated perfusion parameter maps. Magn Reson Med 79:1345-1353, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.
XGC developments for a more efficient XGC-GENE code coupling

NASA Astrophysics Data System (ADS)

Dominski, Julien; Hager, Robert; Ku, Seung-Hoe; Chang, Cs

2017-10-01

In the Exascale Computing Program, the High-Fidelity Whole Device Modeling project initially aims at delivering a tightly-coupled simulation of plasma neoclassical and turbulence dynamics from the core to the edge of the tokamak. To permit such simulations, the gyrokinetic codes GENE and XGC will be coupled together. Numerical efforts are made to improve the numerical schemes agreement in the coupling region. One of the difficulties of coupling those codes together is the incompatibility of their grids. GENE is a continuum grid-based code and XGC is a Particle-In-Cell code using unstructured triangular mesh. A field-aligned filter is thus implemented in XGC. Even if XGC originally had an approximately field-following mesh, this field-aligned filter permits to have a perturbation discretization closer to the one solved in the field-aligned code GENE. Additionally, new XGC gyro-averaging matrices are implemented on a velocity grid adapted to the plasma properties, thus ensuring same accuracy from the core to the edge regions.
IMU-to-Segment Assignment and Orientation Alignment for the Lower Body Using Deep Learning

PubMed Central

2018-01-01

Human body motion analysis based on wearable inertial measurement units (IMUs) receives a lot of attention from both the research community and the and industrial community. This is due to the significant role in, for instance, mobile health systems, sports and human computer interaction. In sensor based activity recognition, one of the major issues for obtaining reliable results is the sensor placement/assignment on the body. For inertial motion capture (joint kinematics estimation) and analysis, the IMU-to-segment (I2S) assignment and alignment are central issues to obtain biomechanical joint angles. Existing approaches for I2S assignment usually rely on hand crafted features and shallow classification approaches (e.g., support vector machines), with no agreement regarding the most suitable features for the assignment task. Moreover, estimating the complete orientation alignment of an IMU relative to the segment it is attached to using a machine learning approach has not been shown in literature so far. This is likely due to the high amount of training data that have to be recorded to suitably represent possible IMU alignment variations. In this work, we propose online approaches for solving the assignment and alignment tasks for an arbitrary amount of IMUs with respect to a biomechanical lower body model using a deep learning architecture and windows of 128 gyroscope and accelerometer data samples. For this, we combine convolutional neural networks (CNNs) for local filter learning with long-short-term memory (LSTM) recurrent networks as well as generalized recurrent units (GRUs) for learning time dynamic features. The assignment task is casted as a classification problem, while the alignment task is casted as a regression problem. In this framework, we demonstrate the feasibility of augmenting a limited amount of real IMU training data with simulated alignment variations and IMU data for improving the recognition/estimation accuracies. With the proposed approaches and final models we achieved 98.57% average accuracy over all segments for the I2S assignment task (100% when excluding left/right switches) and an average median angle error over all segments and axes of 2.91° for the I2S alignment task. PMID:29351262
Three-dimensional dynamic deformation monitoring using a laser-scanning system

NASA Astrophysics Data System (ADS)

Al-Hanbali, Nedal N.; Teskey, William F.

1994-10-01

Non-contact dynamic deformation monitoring (e.g. with a laser scanning system) is very useful in monitoring changes in alignment and changes in size and shape of coupled operating machines. If relative movements between coupled operating machines are large, excessive wear in the machines or unplanned shutdowns due to machinery failure will occur. The purpose of non-contact dynamic deformation monitoring is to identify the causes of large movements and point to remedial action that can be taken to prevent them. The laser scanning system is a laser-based 3D vision system. The system-technique is based on an auto- synchronized triangulation scanning scheme. The system provides accurate, fast, and reliable 3D measurements and can measure objects between 0.5 m to 100 m with a field of view of 40 degree(s) X 50 degree(s). The system is flexible in terms of providing control over the scanned area and depth. The system also provides the user with the intensity image in addition to the depth coded image. This paper reports on the preliminary testing of this system to monitor surface movements and target (point) movements. The monitoring resolution achieved for an operating motorized alignment test rig in the lab was 1 mm for surface movements and 0.50 m for target movements. Raw data manipulation, local calibration, and the method of relating measurements to control points will be discussed. Possibilities for improving the resolution and recommendations for future development will also be presented.
Electronic properties of semiconductor-water interfaces: Predictions from ab-initio molecular dynamics and many-body perturbation theory

NASA Astrophysics Data System (ADS)

Pham, Tuan Anh

2015-03-01

Photoelectrochemical cells offer a promising avenue for hydrogen production from water and sunlight. The efficiency of these devices depends on the electronic structure of the interface between the photoelectrode and liquid water, including the alignment between the semiconductor band edges and the water redox potential. In this talk, we will present the results of first principles calculations of semiconductor-water interfaces that are obtained with a combination of density functional theory (DFT)-based molecular dynamics simulations and many-body perturbation theory (MBPT). First, we will discuss the development of an MBPT approach that is aimed at improving the efficiency and accuracy of existing methodologies while still being applicable to complex heterogeneous interfaces consisting of hundreds of atoms. We will then present studies of the electronic structure of liquid water and aqueous solutions using MBPT, which represent an essential step in establishing a quantitative framework for computing the energy alignment at semiconductor-water interfaces. Finally, using a combination of DFT-based molecular dynamics simulations and MBPT, we will describe the relationship between interfacial structure, electronic properties of semiconductors and their reactivity in aqueous solutions through a number of examples, including functionalized Si surfaces and GaP/InP surfaces in contact with liquid water. T.A.P was supported by the U.S. Department of Energy at the Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344 and by the Lawrence Fellowship Program.
A generalized global alignment algorithm.

PubMed

Huang, Xiaoqiu; Chao, Kun-Mao

2003-01-22

Homologous sequences are sometimes similar over some regions but different over other regions. Homologous sequences have a much lower global similarity if the different regions are much longer than the similar regions. We present a generalized global alignment algorithm for comparing sequences with intermittent similarities, an ordered list of similar regions separated by different regions. A generalized global alignment model is defined to handle sequences with intermittent similarities. A dynamic programming algorithm is designed to compute an optimal general alignment in time proportional to the product of sequence lengths and in space proportional to the sum of sequence lengths. The algorithm is implemented as a computer program named GAP3 (Global Alignment Program Version 3). The generalized global alignment model is validated by experimental results produced with GAP3 on both DNA and protein sequences. The GAP3 program extends the ability of standard global alignment programs to recognize homologous sequences of lower similarity. The GAP3 program is freely available for academic use at http://bioinformatics.iastate.edu/aat/align/align.html.
Online intelligent controllers for an enzyme recovery plant: design methodology and performance.

PubMed

Leite, M S; Fujiki, T L; Silva, F V; Fileti, A M F

2010-12-27

This paper focuses on the development of intelligent controllers for use in a process of enzyme recovery from pineapple rind. The proteolytic enzyme bromelain (EC 3.4.22.4) is precipitated with alcohol at low temperature in a fed-batch jacketed tank. Temperature control is crucial to avoid irreversible protein denaturation. Fuzzy or neural controllers offer a way of implementing solutions that cover dynamic and nonlinear processes. The design methodology and a comparative study on the performance of fuzzy-PI, neurofuzzy, and neural network intelligent controllers are presented. To tune the fuzzy PI Mamdani controller, various universes of discourse, rule bases, and membership function support sets were tested. A neurofuzzy inference system (ANFIS), based on Takagi-Sugeno rules, and a model predictive controller, based on neural modeling, were developed and tested as well. Using a Fieldbus network architecture, a coolant variable speed pump was driven by the controllers. The experimental results show the effectiveness of fuzzy controllers in comparison to the neural predictive control. The fuzzy PI controller exhibited a reduced error parameter (ITAE), lower power consumption, and better recovery of enzyme activity.
Online Intelligent Controllers for an Enzyme Recovery Plant: Design Methodology and Performance

PubMed Central

Leite, M. S.; Fujiki, T. L.; Silva, F. V.; Fileti, A. M. F.

2010-01-01

This paper focuses on the development of intelligent controllers for use in a process of enzyme recovery from pineapple rind. The proteolytic enzyme bromelain (EC 3.4.22.4) is precipitated with alcohol at low temperature in a fed-batch jacketed tank. Temperature control is crucial to avoid irreversible protein denaturation. Fuzzy or neural controllers offer a way of implementing solutions that cover dynamic and nonlinear processes. The design methodology and a comparative study on the performance of fuzzy-PI, neurofuzzy, and neural network intelligent controllers are presented. To tune the fuzzy PI Mamdani controller, various universes of discourse, rule bases, and membership function support sets were tested. A neurofuzzy inference system (ANFIS), based on Takagi-Sugeno rules, and a model predictive controller, based on neural modeling, were developed and tested as well. Using a Fieldbus network architecture, a coolant variable speed pump was driven by the controllers. The experimental results show the effectiveness of fuzzy controllers in comparison to the neural predictive control. The fuzzy PI controller exhibited a reduced error parameter (ITAE), lower power consumption, and better recovery of enzyme activity. PMID:21234106
Nucleotide Sequence of the blaRTG-2 (CARB-5) Gene and Phylogeny of a New Group of Carbenicillinases

PubMed Central

Choury, Daniele; Szajnert, Marie-France; Joly-Guillou, Marie-Laure; Azibi, Kemal; Delpech, Marc; Paul, Gérard

2000-01-01

We determined the nucleotide sequence of the bla gene for the Acinetobacter calcoaceticus β-lactamase previously described as CARB-5. Alignment of the deduced amino acid sequence with those of known β-lactamases revealed that CARB-5 possesses an RTG triad in box VII, as described for the Proteus mirabilis GN79 enzyme, instead of the RSG consensus characteristic of the other carbenicillinases. Phylogenetic studies showed that these RTG enzymes constitute a new, separate group, possibly ancestors of the carbenicillinase family. PMID:10722515
Extremely High Thermal Conductivity of Aligned Carbon Nanotube-Polyethylene Composites.

PubMed

Liao, Quanwen; Liu, Zhichun; Liu, Wei; Deng, Chengcheng; Yang, Nuo

2015-11-10

The ultra-low thermal conductivity of bulk polymers may be enhanced by combining them with high thermal conductivity materials such as carbon nanotubes. Different from random doping, we find that the aligned carbon nanotube-polyethylene composites has a high thermal conductivity by non-equilibrium molecular dynamics simulations. The analyses indicate that the aligned composite not only take advantage of the high thermal conduction of carbon nanotubes, but enhance thermal conduction of polyethylene chains.
QSAR for RNases and theoretic-experimental study of molecular diversity on peptide mass fingerprints of a new Leishmania infantum protein.

PubMed

González-Díaz, Humberto; Dea-Ayuela, María A; Pérez-Montoto, Lázaro G; Prado-Prado, Francisco J; Agüero-Chapín, Guillermín; Bolas-Fernández, Francisco; Vazquez-Padrón, Roberto I; Ubeira, Florencio M

2010-05-01

The toxicity and low success of current treatments for Leishmaniosis determines the search of new peptide drugs and/or molecular targets in Leishmania pathogen species (L. infantum and L. major). For example, Ribonucleases (RNases) are enzymes relevant to several biologic processes; then, theoretical and experimental study of the molecular diversity of Peptide Mass Fingerprints (PMFs) of RNases is useful for drug design. This study introduces a methodology that combines QSAR models, 2D-Electrophoresis (2D-E), MALDI-TOF Mass Spectroscopy (MS), BLAST alignment, and Molecular Dynamics (MD) to explore PMFs of RNases. We illustrate this approach by investigating for the first time the PMFs of a new protein of L. infantum. Here we report and compare new versus old predictive models for RNases based on Topological Indices (TIs) of Markov Pseudo-Folding Lattices. These group of indices called Pseudo-folding Lattice 2D-TIs include: Spectral moments pi ( k )(x,y), Mean Electrostatic potentials xi ( k )(x,y), and Entropy measures theta ( k )(x,y). The accuracy of the models (training/cross-validation) was as follows: xi ( k )(x,y)-model (96.0%/91.7%)>pi ( k )(x,y)-model (84.7/83.3) > theta ( k )(x,y)-model (66.0/66.7). We also carried out a 2D-E analysis of biological samples of L. infantum promastigotes focusing on a 2D-E gel spot of one unknown protein with M<20, 100 and pI <7. MASCOT search identified 20 proteins with Mowse score >30, but not one >52 (threshold value), the higher value of 42 was for a probable DNA-directed RNA polymerase. However, we determined experimentally the sequence of more than 140 peptides. We used QSAR models to predict RNase scores for these peptides and BLAST alignment to confirm some results. We also calculated 3D-folding TIs based on MD experiments and compared 2D versus 3D-TIs on molecular phylogenetic analysis of the molecular diversity of these peptides. This combined strategy may be of interest in drug development or target identification.
Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease

PubMed Central

Wei, Guoxian; Tian, Na; Siezen, Roland; Schuppan, Detlef

2016-01-01

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD. PMID:27469368
A study of the dynamic tire properties over a range of tire constructions

NASA Technical Reports Server (NTRS)

Nybakken, G. H.; Dodge, R. N.; Clark, S. K.

1973-01-01

The dynamic properties of four model aircraft tires of various construction were evaluated experimentally and compared with available theory. The experimental investigation consisted of measuring the cornering force and the self-aligning torque developed by the tires undergoing sinusoidal steering inputs while operating on a small scale, road-wheel tire testing apparatus. The force and moment data from the different tires are compared with both finite- and point-contact patch string theory predictions. In general, agreement between finite contact patch theory and experimental observation is good. A modified string theory is also presented in which coefficients for cornering force and self-aligning torque are determined separately. This theory improves the correspondence between the experimental and analytical data, particularly on tires with relatively high self-aligning torques.

Highly sensitive biofunctionalized mesoporous electrospun TiO(2) nanofiber based interface for biosensing.

PubMed

Mondal, Kunal; Ali, Md Azahar; Agrawal, Ved V; Malhotra, Bansi D; Sharma, Ashutosh

2014-02-26

The surface modified and aligned mesoporous anatase titania nanofiber mats (TiO2-NF) have been fabricated by electrospinning for esterified cholesterol detection by electrochemical technique. The electrospinning and porosity of mesoporous TiO2-NF were controlled by use of polyvinylpyrrolidone (PVP) as a sacrificial carrier polymer in the titanium isopropoxide precursor. The mesoporous TiO2-NF of diameters ranging from 30 to 60 nm were obtained by calcination at 470 °C and partially aligned on a rotating drum collector. The functional groups such as -COOH, -CHO etc. were introduced on TiO2-NF surface via oxygen plasma treatment making the surface hydrophilic. Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) were covalently immobilized on the plasma treated surface of NF (cTiO2-NF) via N-ethyl-N0-(3-dimethylaminopropyl carbodiimide) and N-hydroxysuccinimide (EDC-NHS) chemistry. The high mesoporosity (∼61%) of the fibrous film allowed enhanced loading of the enzyme molecules in the TiO2-NF mat. The ChEt-ChOx/cTiO2-NF-based bioelectrode was used to detect esterified cholesterol using electrochemical technique. The high aspect ratio, surface area of aligned TiO2-NF showed excellent voltammetric and catalytic response resulting in improved detection limit (0.49 mM). The results of response studies of this biosensor show excellent sensitivity (181.6 μA/mg dL(-1)/cm(2)) and rapid detection (20 s). This proposed strategy of biomolecule detection is thus a promising platform for the development of miniaturized device for biosensing applications.
Quantum Mechanical Modeling: A Tool for the Understanding of Enzyme Reactions

PubMed Central

Náray-Szabó, Gábor; Oláh, Julianna; Krámos, Balázs

2013-01-01

Most enzyme reactions involve formation and cleavage of covalent bonds, while electrostatic effects, as well as dynamics of the active site and surrounding protein regions, may also be crucial. Accordingly, special computational methods are needed to provide an adequate description, which combine quantum mechanics for the reactive region with molecular mechanics and molecular dynamics describing the environment and dynamic effects, respectively. In this review we intend to give an overview to non-specialists on various enzyme models as well as established computational methods and describe applications to some specific cases. For the treatment of various enzyme mechanisms, special approaches are often needed to obtain results, which adequately refer to experimental data. As a result of the spectacular progress in the last two decades, most enzyme reactions can be quite precisely treated by various computational methods. PMID:24970187
Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

PubMed

Vieille, C; Zeikus, G J

2001-03-01

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.
Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

PubMed Central

Vieille, Claire; Zeikus, Gregory J.

2001-01-01

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described. PMID:11238984
Miniaturized GPS/MEMS IMU integrated board

NASA Technical Reports Server (NTRS)

Lin, Ching-Fang (Inventor)

2012-01-01

This invention documents the efforts on the research and development of a miniaturized GPS/MEMS IMU integrated navigation system. A miniaturized GPS/MEMS IMU integrated navigation system is presented; Laser Dynamic Range Imager (LDRI) based alignment algorithm for space applications is discussed. Two navigation cameras are also included to measure the range and range rate which can be integrated into the GPS/MEMS IMU system to enhance the navigation solution.
Radial SI latches vibration test data review

NASA Technical Reports Server (NTRS)

Harrison, P. M.; Smith, J. L.

1984-01-01

Dynamic testing of the Space Telescope Scientific Instrument Radial Latches was performed as specified by the designated test criteria. No structural failures were observed during the test. The alignment stability of the instrument simulator was within required tolerances after testing. Particulates were discovered around the latch bases, after testing, due to wearing at the B and C latch interface surfaces. This report covers criteria derivation, testing, and test results.
Relaxation processes in a low-order three-dimensional magnetohydrodynamics model

NASA Technical Reports Server (NTRS)

Stribling, Troy; Matthaeus, William H.

1991-01-01

The time asymptotic behavior of a Galerkin model of 3D magnetohydrodynamics (MHD) has been interpreted using the selective decay and dynamic alignment relaxation theories. A large number of simulations has been performed that scan a parameter space defined by the rugged ideal invariants, including energy, cross helicity, and magnetic helicity. It is concluded that time asymptotic state can be interpreted as a relaxation to minimum energy. A simple decay model, based on absolute equilibrium theory, is found to predict a mapping of initial onto time asymptotic states, and to accurately describe the long time behavior of the runs when magnetic helicity is present. Attention is also given to two processes, operating on time scales shorter than selective decay and dynamic alignment, in which the ratio of kinetic to magnetic energy relaxes to values 0(1). The faster of the two processes takes states initially dominant in magnetic energy to a state of near-equipartition between kinetic and magnetic energy through power law growth of kinetic energy. The other process takes states initially dominant in kinetic energy to the near-equipartitioned state through exponential growth of magnetic energy.
Ultrasound monitoring of inter-knee distances during gait.

PubMed

Lai, Daniel T H; Wrigley, Tim V; Palaniswami, M

2009-01-01

Knee osteoarthritis is an extremely common, debilitating disease associated with pain and loss of function. There is considerable interest in monitoring lower limb alignment due to its close association with joint overload leading to disease progression. The effects of gait modifications that can lower joint loading are of particular interest. Here we describe an ultrasound-based system for monitoring an important aspect of dynamic lower limb alignment, the inter-knee distance during walking. Monitoring this gait parameter should facilitate studies in reducing knee loading, a primary risk factor of knee osteoarthritis progression. The portable device is composed of an ultrasound sensor connected to an Intel iMote2 equipped with Bluetooth wireless capability. Static tests and calibration results show that the sensor possesses an effective beam envelope of 120 degrees, with maximum distance errors of 10% at the envelope edges. Dynamic walking trials reveal close correlation of inter-knee distance trends between that measured by an optical system (Optotrak Certus NDI) and the sensor device. The maximum average root mean square error was found to be 1.46 cm. Future work will focus on improving the accuracy of the device.
A priori testing of subgrid-scale models for large-eddy simulation of the atmospheric boundary layer

NASA Astrophysics Data System (ADS)

Juneja, Anurag; Brasseur, James G.

1996-11-01

Subgrid-scale models are generally developed assuming homogeneous isotropic turbulence with the filter cutoff lying in the inertial range. In the surface layer and capping inversion regions of the atmospheric boundary layer, the turbulence is strongly anisotropic and, in general, influenced by both buoyancy and shear. Furthermore, the integral scale motions are under-resolved in these regions. Herein we perform direct numerical simulations of shear and buoyancy-generated homogeneous anisotropic turbulence to compute and analyze the actual subgrid-resolved-scale (SGS-RS) dynamics as the filter cutoff moves into the energy-containing scales. These are compared with the SGS-RS dynamics predicted by Smagorinsky-based models with a focus on motivating improved closures. We find that, in general, the underlying assumption of such models, that the anisotropic part of the subgrid stress tensor be aligned with the resolved strain rate tensor, is a poor approximation. Similarly, we find poor alignment between the actual and predicted stress divergence, and find low correlations between the actual and modeled subgrid-scale contribution to the pressure and pressure gradient. Details will be given in the talk.
A significant role of non-thermal equilibrated electrons in the formation of deleterious complex DNA damage.

PubMed

Kai, Takeshi; Yokoya, Akinari; Ukai, Masatoshi; Fujii, Kentaro; Toigawa, Tomohiro; Watanabe, Ritsuko

2018-01-24

Although most of the radiation damage to genomic DNA could be rendered harmless using repair enzymes in a living cell, a certain fraction of the damage is persistent resulting in serious genetic effects, such as mutation induction. In order to understand the mechanisms of the deleterious DNA damage formation in terms of its earliest physical stage at the radiation track end, dynamics of low energy electrons and their thermalization processes around DNA molecules were investigated using a dynamic Monte Carlo code. The primary incident (1 keV) electrons multiply collide within 1 nm (equivalent to three DNA-base-pairs, 3bp) and generate secondary electrons which show non-Gaussian and non-thermal equilibrium distributions within 300 fs. On the other hand, the secondary electrons are mainly distributed within approximately 10 nm from their parent cations although approximately 5% of the electrons are localized within 1 nm of the cations owing to the interaction of their Coulombic fields. The mean electron energy is 0.7 eV; however, more than 10% of the electrons fall into a much lower-energy region than 0.1 eV at 300 fs. These results indicate that pre-hydrated electrons are formed from the extremely decelerated electrons over a few nm from the cations. DNA damage sites comprising multiple nucleobase lesions or single strand breaks can therefore be formed by multiple collisions of these electrons within 3bp. This multiple damage site is hardly processed by base excision repair enzymes. However, pre-hydrated electrons can also be produced resulting in an additional base lesion (or a strand break) more than 3bp away from the multi-damage site. These damage sites may be finally converted into a double strand break (DSB) when base excision enzymes process the additional base lesions. This DSB includes another base lesion(s) at their termini, and may introduce miss-rejoining by DSB repair enzymes, and hence may result in biological effects such as mutation in surviving cells.
An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

PubMed

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F; Cohen, Itay; Henin, Rachel D; Hockla, Alexandra; Soares, Alexei S; Papo, Niv; Caulfield, Thomas R; Radisky, Evette S

2016-12-16

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Conformational diversity and computational enzyme design

PubMed Central

Lassila, Jonathan K.

2010-01-01

The application of computational protein design methods to the design of enzyme active sites offers potential routes to new catalysts and new reaction specificities. Computational design methods have typically treated the protein backbone as a rigid structure for the sake of computational tractability. However, this fixed-backbone approximation introduces its own special challenges for enzyme design and it contrasts with an emerging picture of natural enzymes as dynamic ensembles with multiple conformations and motions throughout a reaction cycle. This review considers the impact of conformational variation and dynamics on computational enzyme design and it highlights new approaches to addressing protein conformational diversity in enzyme design including recent advances in multistate design, backbone flexibility, and computational library design. PMID:20829099
How Accurate Are Transition States from Simulations of Enzymatic Reactions?

PubMed Central

2015-01-01

The rate expression of traditional transition state theory (TST) assumes no recrossing of the transition state (TS) and thermal quasi-equilibrium between the ground state and the TS. Currently, it is not well understood to what extent these assumptions influence the nature of the activated complex obtained in traditional TST-based simulations of processes in the condensed phase in general and in enzymes in particular. Here we scrutinize these assumptions by characterizing the TSs for hydride transfer catalyzed by the enzyme Escherichia coli dihydrofolate reductase obtained using various simulation approaches. Specifically, we compare the TSs obtained with common TST-based methods and a dynamics-based method. Using a recently developed accurate hybrid quantum mechanics/molecular mechanics potential, we find that the TST-based and dynamics-based methods give considerably different TS ensembles. This discrepancy, which could be due equilibrium solvation effects and the nature of the reaction coordinate employed and its motion, raises major questions about how to interpret the TSs determined by common simulation methods. We conclude that further investigation is needed to characterize the impact of various TST assumptions on the TS phase-space ensemble and on the reaction kinetics. PMID:24860275
Assessing the statistical robustness of inter- and intra-basinal carbon isotope chemostratigraphic correlation

NASA Astrophysics Data System (ADS)

Hay, C.; Creveling, J. R.; Huybers, P. J.

2016-12-01

Excursions in the stable carbon isotopic composition of carbonate rocks (δ13Ccarb) can facilitate correlation of Precambrian and Phanerozoic sedimentary successions at a higher temporal resolution than radiometric and biostratigraphic frameworks typically afford. Within the bounds of litho- and biostratigraphic constraints, stratigraphers often correlate isotopic patterns between distant stratigraphic sections through visual alignment of local maxima and minima of isotopic values. The reproducibility of this method can prove challenging and, thus, evaluating the statistical robustness of intrabasinal composite carbon isotope curves, and global correlations to these reference curves, remains difficult. To assess the reproducibility of stratigraphic alignment of δ13Ccarb data, and correlations between carbon isotope excursions, we employ a numerical dynamic time warping methodology that stretches and squeezes the time axis of a record to obtain an optimal correlation (in a least-squares sense) between time-uncertain series of data. In particular, we assess various alignments between series of Early Cambrian δ13Ccarb data with respect to plausible matches. We first show that an alignment of these records obtained visually, and published previously, is broadly reproducible using dynamic time warping. Alternative alignments with similar goodness of fits are also obtainable, and their stratigraphic plausibility are discussed. This approach should be generalizable to an algorithm for the purposes of developing a library of plausible alignments between multiple time-uncertain stratigraphic records.
Comparison of Three Ionic Liquid-Tolerant Cellulases by Molecular Dynamics

PubMed Central

Jaeger, Vance; Burney, Patrick; Pfaendtner, Jim

2015-01-01

We have employed molecular dynamics to investigate the differences in ionic liquid tolerance among three distinct family 5 cellulases from Trichoderma viride, Thermogata maritima, and Pyrococcus horikoshii. Simulations of the three cellulases were conducted at a range of temperatures in various binary mixtures of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate with water. Our analysis demonstrates that the effects of ionic liquids on the enzymes vary in each individual case from local structural disturbances to loss of much of one of the enzyme’s secondary structure. Enzymes with more negatively charged surfaces tend to resist destabilization by ionic liquids. Specific and unique structural changes in the enzymes are induced by the presence of ionic liquids. Disruption of the secondary structure, changes in dynamical motion, and local changes in the binding pocket are observed in less tolerant enzymes. Ionic-liquid-induced denaturation of one of the enzymes is indicated over the 500 ns timescale. In contrast, the most tolerant cellulase behaves similarly in water and in ionic-liquid-containing mixtures. Unlike the heuristic approaches that attempt to predict enzyme stability using macroscopic properties, molecular dynamics allows us to predict specific atomic-level structural and dynamical changes in an enzyme’s behavior induced by ionic liquids and other mixed solvents. Using these insights, we propose specific experimentally testable hypotheses regarding the origin of activity loss for each of the systems investigated in this study. PMID:25692593
CombAlign: a code for generating a one-to-many sequence alignment from a set of pairwise structure-based sequence alignments.

PubMed

Zhou, Carol L Ecale

2015-01-01

In order to better define regions of similarity among related protein structures, it is useful to identify the residue-residue correspondences among proteins. Few codes exist for constructing a one-to-many multiple sequence alignment derived from a set of structure or sequence alignments, and a need was evident for creating such a tool for combining pairwise structure alignments that would allow for insertion of gaps in the reference structure. This report describes a new Python code, CombAlign, which takes as input a set of pairwise sequence alignments (which may be structure based) and generates a one-to-many, gapped, multiple structure- or sequence-based sequence alignment (MSSA). The use and utility of CombAlign was demonstrated by generating gapped MSSAs using sets of pairwise structure-based sequence alignments between structure models of the matrix protein (VP40) and pre-small/secreted glycoprotein (sGP) of Reston Ebolavirus and the corresponding proteins of several other filoviruses. The gapped MSSAs revealed structure-based residue-residue correspondences, which enabled identification of structurally similar versus differing regions in the Reston proteins compared to each of the other corresponding proteins. CombAlign is a new Python code that generates a one-to-many, gapped, multiple structure- or sequence-based sequence alignment (MSSA) given a set of pairwise sequence alignments (which may be structure based). CombAlign has utility in assisting the user in distinguishing structurally conserved versus divergent regions on a reference protein structure relative to other closely related proteins. CombAlign was developed in Python 2.6, and the source code is available for download from the GitHub code repository.
A QM/MM Metadynamics Study of the Direct Decarboxylation Mechanism for Orotidine-5'-monophosphate Decarboxylase using Two Different QM Regions: Acceleration too Small to Explain Rate of Enzyme Catalysis

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

Stanton, Courtney; Kuo, I-F W.; Mundy, Christopher J.

2007-11-01

Despite decades of study, the mechanism of orotidine-5'-monophosphate decarboxylase (ODCase) remains unresolved. A computational investigation of the direct decarboxylation mechanism has been performed using mixed quantum mechanical/molecular mechanical (QM/MM) dynamics simulations. The study was performed with the program CP2K that integrates classical dynamics and ab initio dynamics based on the Born-Oppenheimer approach. Two different QM regions were explored. It was found that the size of the QM region has a dramatic effect on the calculated reaction barrier. The free energy barriers for decarboxylation of orotidine-5'-monophosphate (OMP) in solution and in the enzyme were determined with the metadynamics method to bemore » 40 kcal/mol and 33 kcal/mol, respectively. The calculated change in activation free energy (ΔΔG±) on going from solution to the enzyme is therefore -7 kcal/mol, far less than the experimental change of -23 kcal/mol (for kcat/kuncat Radzicka, A.; Wolfenden, R., Science. 1995, 267, 90-92). These results do not support the direct decarboxylation mechanism in the enzyme. Funding was provided by the University of California Lawrence Livermore National Laboratory (LLNL) and the National Institutes of Health (NIH). Part of this work was performed under the auspices of the U.S. Department of Energy by LLNL under contract No. W-7405-Eng-48. Computer resources were provided by Livermore Computing.« less
A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase

PubMed Central

Bae, Sung-Hun; Duggan, Brendan M.; Benkovic, Stephen J.; Dyson, H. Jane; Wright, Peter E

2013-01-01

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in E.coli dihydrofolate reductase results in up to a 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In the present study, NMR relaxation experiments are used to demonstrate that dynamics on the ps-ns and μs-ms timescales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, ps-ns timescale dynamics are decreased in the FG loop (containing the mutated residue 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and NADP+ bound, assume an occluded ground state conformation, and we do not observe sampling of a higher energy closed conformation by 15N R2 relaxation dispersion. This is highly significant, since it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs μs - ms timescale fluctuations that have been implicated in product release from the wild type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis. PMID:23758161
Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases*

PubMed Central

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

2013-01-01

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

PubMed Central

Brünger, A T; Brooks, C L; Karplus, M

1985-01-01

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state. Images PMID:3866234

Mediolateral force distribution at the knee joint shifts across activities and is driven by tibiofemoral alignment.

PubMed

Kutzner, I; Bender, A; Dymke, J; Duda, G; von Roth, P; Bergmann, G

2017-06-01

Tibiofemoral alignment is important to determine the rate of progression of osteoarthritis and implant survival after total knee arthroplasty (TKA). Normally, surgeons aim for neutral tibiofemoral alignment following TKA, but this has been questioned in recent years. The aim of this study was to evaluate whether varus or valgus alignment indeed leads to increased medial or lateral tibiofemoral forces during static and dynamic weight-bearing activities. Tibiofemoral contact forces and moments were measured in nine patients with instrumented knee implants. Medial force ratios were analysed during nine daily activities, including activities with single-limb support (e.g. walking) and double-limb support (e.g. knee bend). Hip-knee-ankle angles in the frontal plane were analysed using full-leg coronal radiographs. The medial force ratio strongly correlated with the tibiofemoral alignment in the static condition of one-legged stance (R² = 0.88) and dynamic single-limb loading (R² = 0.59) with varus malalignment leading to increased medial force ratios of up to 88%. In contrast, the correlation between leg alignment and magnitude of medial compartment force was much less pronounced. A lateral shift of force occurred during activities with double-limb support and higher knee flexion angles. The medial force ratio depends on both the tibiofemoral alignment and the nature of the activity involved. It cannot be generalised to a single value. Higher medial ratios during single-limb loading are associated with varus malalignment in TKA. The current trend towards a 'constitutional varus' after joint replacement, in terms of overall tibiofemoral alignment, should be considered carefully with respect to the increased medial force ratio. Cite this article: Bone Joint J 2017;99-B:779-87. ©2017 The British Editorial Society of Bone & Joint Surgery.
Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

PubMed Central

Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

2017-01-01

Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410
A New Concept to Reveal Protein Dynamics Based on Energy Dissipation

PubMed Central

Ma, Cheng-Wei; Xiu, Zhi-Long; Zeng, An-Ping

2011-01-01

Protein dynamics is essential for its function, especially for intramolecular signal transduction. In this work we propose a new concept, energy dissipation model, to systematically reveal protein dynamics upon effector binding and energy perturbation. The concept is applied to better understand the intramolecular signal transduction during allostery of enzymes. The E. coli allosteric enzyme, aspartokinase III, is used as a model system and special molecular dynamics simulations are designed and carried out. Computational results indicate that the number of residues affected by external energy perturbation (i.e. caused by a ligand binding) during the energy dissipation process shows a sigmoid pattern. Using the two-state Boltzmann equation, we define two parameters, the half response time and the dissipation rate constant, which can be used to well characterize the energy dissipation process. For the allostery of aspartokinase III, the residue response time indicates that besides the ACT2 signal transduction pathway, there is another pathway between the regulatory site and the catalytic site, which is suggested to be the β15-αK loop of ACT1. We further introduce the term “protein dynamical modules” based on the residue response time. Different from the protein structural modules which merely provide information about the structural stability of proteins, protein dynamical modules could reveal protein characteristics from the perspective of dynamics. Finally, the energy dissipation model is applied to investigate E. coli aspartokinase III mutations to better understand the desensitization of product feedback inhibition via allostery. In conclusion, the new concept proposed in this paper gives a novel holistic view of protein dynamics, a key question in biology with high impacts for both biotechnology and biomedicine. PMID:22022616
Development and application of a modified dynamic time warping algorithm (DTW-S) to analyses of primate brain expression time series

PubMed Central

2011-01-01

Background Comparing biological time series data across different conditions, or different specimens, is a common but still challenging task. Algorithms aligning two time series represent a valuable tool for such comparisons. While many powerful computation tools for time series alignment have been developed, they do not provide significance estimates for time shift measurements. Results Here, we present an extended version of the original DTW algorithm that allows us to determine the significance of time shift estimates in time series alignments, the DTW-Significance (DTW-S) algorithm. The DTW-S combines important properties of the original algorithm and other published time series alignment tools: DTW-S calculates the optimal alignment for each time point of each gene, it uses interpolated time points for time shift estimation, and it does not require alignment of the time-series end points. As a new feature, we implement a simulation procedure based on parameters estimated from real time series data, on a series-by-series basis, allowing us to determine the false positive rate (FPR) and the significance of the estimated time shift values. We assess the performance of our method using simulation data and real expression time series from two published primate brain expression datasets. Our results show that this method can provide accurate and robust time shift estimates for each time point on a gene-by-gene basis. Using these estimates, we are able to uncover novel features of the biological processes underlying human brain development and maturation. Conclusions The DTW-S provides a convenient tool for calculating accurate and robust time shift estimates at each time point for each gene, based on time series data. The estimates can be used to uncover novel biological features of the system being studied. The DTW-S is freely available as an R package TimeShift at http://www.picb.ac.cn/Comparative/data.html. PMID:21851598
Development and application of a modified dynamic time warping algorithm (DTW-S) to analyses of primate brain expression time series.

PubMed

Yuan, Yuan; Chen, Yi-Ping Phoebe; Ni, Shengyu; Xu, Augix Guohua; Tang, Lin; Vingron, Martin; Somel, Mehmet; Khaitovich, Philipp

2011-08-18

Comparing biological time series data across different conditions, or different specimens, is a common but still challenging task. Algorithms aligning two time series represent a valuable tool for such comparisons. While many powerful computation tools for time series alignment have been developed, they do not provide significance estimates for time shift measurements. Here, we present an extended version of the original DTW algorithm that allows us to determine the significance of time shift estimates in time series alignments, the DTW-Significance (DTW-S) algorithm. The DTW-S combines important properties of the original algorithm and other published time series alignment tools: DTW-S calculates the optimal alignment for each time point of each gene, it uses interpolated time points for time shift estimation, and it does not require alignment of the time-series end points. As a new feature, we implement a simulation procedure based on parameters estimated from real time series data, on a series-by-series basis, allowing us to determine the false positive rate (FPR) and the significance of the estimated time shift values. We assess the performance of our method using simulation data and real expression time series from two published primate brain expression datasets. Our results show that this method can provide accurate and robust time shift estimates for each time point on a gene-by-gene basis. Using these estimates, we are able to uncover novel features of the biological processes underlying human brain development and maturation. The DTW-S provides a convenient tool for calculating accurate and robust time shift estimates at each time point for each gene, based on time series data. The estimates can be used to uncover novel biological features of the system being studied. The DTW-S is freely available as an R package TimeShift at http://www.picb.ac.cn/Comparative/data.html.
Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

PubMed

Horowitz, Scott; Dirk, Lynnette M A; Yesselman, Joseph D; Nimtz, Jennifer S; Adhikari, Upendra; Mehl, Ryan A; Scheiner, Steve; Houtz, Robert L; Al-Hashimi, Hashim M; Trievel, Raymond C

2013-10-16

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.
Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes

PubMed Central

Carter, Ashley R.; Seaberg, Maasa H.; Fan, Hsiu-Fang; Sun, Gang; Wilds, Christopher J.; Li, Hung-Wen; Perkins, Thomas T.

2016-01-01

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2–4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD–DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5′ strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket. PMID:27220465
Self-Alignment MEMS IMU Method Based on the Rotation Modulation Technique on a Swing Base

PubMed Central

Chen, Zhiyong; Yang, Haotian; Wang, Chengbin; Lin, Zhihui; Guo, Meifeng

2018-01-01

The micro-electro-mechanical-system (MEMS) inertial measurement unit (IMU) has been widely used in the field of inertial navigation due to its small size, low cost, and light weight, but aligning MEMS IMUs remains a challenge for researchers. MEMS IMUs have been conventionally aligned on a static base, requiring other sensors, such as magnetometers or satellites, to provide auxiliary information, which limits its application range to some extent. Therefore, improving the alignment accuracy of MEMS IMU as much as possible under swing conditions is of considerable value. This paper proposes an alignment method based on the rotation modulation technique (RMT), which is completely self-aligned, unlike the existing alignment techniques. The effect of the inertial sensor errors is mitigated by rotating the IMU. Then, inertial frame-based alignment using the rotation modulation technique (RMT-IFBA) achieved coarse alignment on the swing base. The strong tracking filter (STF) further improved the alignment accuracy. The performance of the proposed method was validated with a physical experiment, and the results of the alignment showed that the standard deviations of pitch, roll, and heading angle were 0.0140°, 0.0097°, and 0.91°, respectively, which verified the practicality and efficacy of the proposed method for the self-alignment of the MEMS IMU on a swing base. PMID:29649150
Allosteric dynamics of SAMHD1 studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Patra, K. K.; Bhattacharya, A.; Bhattacharya, S.

2016-10-01

SAMHD1 is a human cellular enzyme that blocks HIV-1 infection in myeloid cells and non-cycling CD4+T cells. The enzyme is an allosterically regulated triphosphohydrolase that modulates the level of cellular dNTP. The virus restriction is attributed to the lowering of the pool of dNTP in the cell to a point where reverse-transcription is impaired. Mutations in SAMHD1 are also implicated in Aicardi-Goutieres syndrome. A mechanistic understanding of the allosteric activation of the enzyme is still elusive. We have performed molecular dynamics simulations to examine the allosteric site dynamics of the protein and to examine the connection between the stability of the tetrameric complex and the Allosite occupancy.
Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors.

PubMed

Liu, Jia; Yang, Qihua; Li, Can

2015-09-18

Biocatalysis with immobilized enzymes as catalysts holds enormous promise in developing more efficient and sustainable processes for the synthesis of fine chemicals, chiral pharmaceuticals and biomass feedstocks. Despite the appealing potentials, nowadays the industrial-scale application of biocatalysts is still quite modest in comparison with that of traditional chemical catalysts. A critical issue is that the catalytic performance of enzymes, the sophisticated and vulnerable catalytic machineries, strongly depends on their intracellular working environment; however the working circumstances provided by the support matrix are radically different from those in cells. This often leads to various adverse consequences on enzyme conformation and dynamic properties, consequently decreasing the overall performance of immobilized enzymes with regard to their activity, selectivity and stability. Engineering enzyme catalysis in support nanopores by mimicking the physiological milieu of enzymes in vivo and investigating how the interior microenvironment of nanopores imposes an influence on enzyme behaviors in vitro are of paramount significance to modify and improve the catalytic functions of immobilized enzymes. In this feature article, we have summarized the recent advances in mimicking the working environment and working patterns of intracellular enzymes in nanopores of mesoporous silica-based supports. Especially, we have demonstrated that incorporation of polymers into silica nanopores could be a valuable approach to create the biomimetic microenvironment for enzymes in the immobilized state.
Correction of static axial alignment in children with knee varus or valgus deformities through guided growth: Does it also correct dynamic frontal plane moments during walking?

PubMed

Böhm, Harald; Stief, Felix; Sander, Klaus; Hösl, Matthias; Döderlein, Leonhard

2015-09-01

Malaligned knees are predisposed to the development and progression of unicompartmental degenerations because of the excessive load placed on one side of the knee. Therefore, guided growth in skeletally immature patients is recommended. Indication for correction of varus/valgus deformities are based on static weight bearing radiographs. However, the dynamic knee abduction moment during walking showed only a weak correlation to malalignment determined by static radiographs. Therefore, the aim of the study was to measure the effects of guided growth on the normalization of frontal plane knee joint moments during walking. 15 legs of 8 patients (11-15 years) with idiopathic axial varus or valgus malalignment were analyzed. 16 typically developed peers served as controls. Instrumented gait analysis and clinical assessment were performed the day before implantation and explantation of eight-plates. Correlation between static mechanical tibiofemoral axis angle (MAA) and dynamic frontal plane knee joint moments and their change by guided growth were performed. The changes in dynamic knee moment in the frontal plane following guided growth showed high and significant correlation to the changes in static MAA (R=0.97, p<0.001). Contrary to the correlation of the changes, there was no correlation between static and dynamic measures in both sessions. In consequence two patients that had a natural knee moment before treatment showed a more pathological one after treatment. In conclusion, the changes in the dynamic load situation during walking can be predicted from the changes in static alignment. If pre-surgical gait analysis reveals a natural load situation, despite a static varus or valgus deformity, the intervention must be critically discussed. Copyright © 2015 Elsevier B.V. All rights reserved.
Molecular and catalytic properties of 2,4'-dihydroxyacetophenone dioxygenase from Burkholderia sp. AZ11.

PubMed

Enya, Mayu; Aoyagi, Keiko; Hishikawa, Yoshihiro; Yoshimura, Azusa; Mitsukura, Koichi; Maruyama, Kiyofumi

2012-01-01

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 µM and 6.28 µmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.
Efficient reductive amination process for enantioselective synthesis of L-phosphinothricin applying engineered glutamate dehydrogenase.

PubMed

Yin, Xinjian; Wu, Jianping; Yang, Lirong

2018-05-01

The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, PpGluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.
Vector analysis of ecoenzyme activities reveal constraints on coupled C, N and P dynamics

EPA Science Inventory

We developed a quantitative method for estimating resource allocation strategies of microbial communities based on the proportional activities of four, key extracellular enzymes, 1,4-ß-glucosidase (BG), leucine amino-peptidase (LAP), 1,4-ß-N-acetylglucosaminidase (NAG...
Mediation of donor–acceptor distance in an enzymatic methyl transfer reaction

PubMed Central

Zhang, Jianyu; Kulik, Heather J.; Martinez, Todd J.; Klinman, Judith P.

2015-01-01

Enzymatic methyl transfer, catalyzed by catechol-O-methyltransferase (COMT), is investigated using binding isotope effects (BIEs), time-resolved fluorescence lifetimes, Stokes shifts, and extended graphics processing unit (GPU)-based quantum mechanics/molecular mechanics (QM/MM) approaches. The WT enzyme is compared with mutants at Tyr68, a conserved residue that is located behind the reactive sulfur of cofactor. Small (>1) BIEs are observed for an S-adenosylmethionine (AdoMet)-binary and abortive ternary complex containing 8-hydroxyquinoline, and contrast with previously reported inverse (<1) kinetic isotope effects (KIEs). Extended GPU-based computational studies of a ternary complex containing catecholate show a clear trend in ground state structures, from noncanonical bond lengths for WT toward solution values with mutants. Structural and dynamical differences that are sensitive to Tyr68 have also been detected using time-resolved Stokes shift measurements and molecular dynamics. These experimental and computational results are discussed in the context of active site compaction that requires an ionization of substrate within the enzyme ternary complex. PMID:26080432
Predicting areas of sustainable error growth in quasigeostrophic flows using perturbation alignment properties

NASA Astrophysics Data System (ADS)

Rivière, G.; Hua, B. L.

2004-10-01

A new perturbation initialization method is used to quantify error growth due to inaccuracies of the forecast model initial conditions in a quasigeostrophic box ocean model describing a wind-driven double gyre circulation. This method is based on recent analytical results on Lagrangian alignment dynamics of the perturbation velocity vector in quasigeostrophic flows. More specifically, it consists in initializing a unique perturbation from the sole knowledge of the control flow properties at the initial time of the forecast and whose velocity vector orientation satisfies a Lagrangian equilibrium criterion. This Alignment-based Initialization method is hereafter denoted as the AI method.In terms of spatial distribution of the errors, we have compared favorably the AI error forecast with the mean error obtained with a Monte-Carlo ensemble prediction. It is shown that the AI forecast is on average as efficient as the error forecast initialized with the leading singular vector for the palenstrophy norm, and significantly more efficient than that for total energy and enstrophy norms. Furthermore, a more precise examination shows that the AI forecast is systematically relevant for all control flows whereas the palenstrophy singular vector forecast leads sometimes to very good scores and sometimes to very bad ones.A principal component analysis at the final time of the forecast shows that the AI mode spatial structure is comparable to that of the first eigenvector of the error covariance matrix for a "bred mode" ensemble. Furthermore, the kinetic energy of the AI mode grows at the same constant rate as that of the "bred modes" from the initial time to the final time of the forecast and is therefore characterized by a sustained phase of error growth. In this sense, the AI mode based on Lagrangian dynamics of the perturbation velocity orientation provides a rationale of the "bred mode" behavior.
Beam/seam alignment control for electron beam welding

DOEpatents

Burkhardt, Jr., James H.; Henry, J. James; Davenport, Clyde M.

1980-01-01

This invention relates to a dynamic beam/seam alignment control system for electron beam welds utilizing video apparatus. The system includes automatic control of workpiece illumination, near infrared illumination of the workpiece to limit the range of illumination and camera sensitivity adjustment, curve fitting of seam position data to obtain an accurate measure of beam/seam alignment, and automatic beam detection and calculation of the threshold beam level from the peak beam level of the preceding video line to locate the beam or seam edges.
Isosteric And Non-Isosteric Base Pairs In RNA Motifs: Molecular Dynamics And Bioinformatics Study Of The Sarcin-Ricin Internal Loop

PubMed Central

Havrila, Marek; Réblová, Kamila; Zirbel, Craig L.; Leontis, Neocles B.; Šponer, Jiří

2013-01-01

The Sarcin-Ricin RNA motif (SR motif) is one of the most prominent recurrent RNA building blocks that occurs in many different RNA contexts and folds autonomously, i.e., in a context-independent manner. In this study, we combined bioinformatics analysis with explicit-solvent molecular dynamics (MD) simulations to better understand the relation between the RNA sequence and the evolutionary patterns of SR motif. SHAPE probing experiment was also performed to confirm fidelity of MD simulations. We identified 57 instances of the SR motif in a non-redundant subset of the RNA X-ray structure database and analyzed their basepairing, base-phosphate, and backbone-backbone interactions. We extracted sequences aligned to these instances from large ribosomal RNA alignments to determine frequency of occurrence for different sequence variants. We then used a simple scoring scheme based on isostericity to suggest 10 sequence variants with highly variable expected degree of compatibility with the SR motif 3D structure. We carried out MD simulations of SR motifs with these base substitutions. Non isosteric base substitutions led to unstable structures, but so did isosteric substitutions which were unable to make key base-phosphate interactions. MD technique explains why some potentially isosteric SR motifs are not realized during evolution. We also found that inability to form stable cWW geometry is an important factor in case of the first base pair of the flexible region of the SR motif. Comparison of structural, bioinformatics, SHAPE probing and MD simulation data reveals that explicit solvent MD simulations neatly reflect viability of different sequence variants of the SR motif. Thus, MD simulations can efficiently complement bioinformatics tools in studies of conservation patterns of RNA motifs and provide atomistic insight into the role of their different signature interactions. PMID:24144333
Phylogenic inference using alignment-free methods for applications in microbial community surveys using 16s rRNA gene

PubMed Central

2017-01-01

The diversity of microbiota is best explored by understanding the phylogenetic structure of the microbial communities. Traditionally, sequence alignment has been used for phylogenetic inference. However, alignment-based approaches come with significant challenges and limitations when massive amounts of data are analyzed. In the recent decade, alignment-free approaches have enabled genome-scale phylogenetic inference. Here we evaluate three alignment-free methods: ACS, CVTree, and Kr for phylogenetic inference with 16s rRNA gene data. We use a taxonomic gold standard to compare the accuracy of alignment-free phylogenetic inference with that of common microbiome-wide phylogenetic inference pipelines based on PyNAST and MUSCLE alignments with FastTree and RAxML. We re-simulate fecal communities from Human Microbiome Project data to evaluate the performance of the methods on datasets with properties of real data. Our comparisons show that alignment-free methods are not inferior to alignment-based methods in giving accurate and robust phylogenic trees. Moreover, consensus ensembles of alignment-free phylogenies are superior to those built from alignment-based methods in their ability to highlight community differences in low power settings. In addition, the overall running times of alignment-based and alignment-free phylogenetic inference are comparable. Taken together our empirical results suggest that alignment-free methods provide a viable approach for microbiome-wide phylogenetic inference. PMID:29136663
Rigid Facial Motion Influences Featural, But Not Holistic, Face Processing

PubMed Central

Xiao, Naiqi; Quinn, Paul C.; Ge, Liezhong; Lee, Kang

2012-01-01

We report three experiments in which we investigated the effect of rigid facial motion on face processing. Specifically, we used the face composite effect to examine whether rigid facial motion influences primarily featural or holistic processing of faces. In Experiments 1, 2, and 3, participants were first familiarized with dynamic displays in which a target face turned from one side to another; then at test, participants judged whether the top half of a composite face (the top half of the target face aligned or misaligned with the bottom half of a foil face) belonged to the target face. We compared performance in the dynamic condition to various static control conditions in Experiments 1, 2, and 3, which differed from each other in terms of the display order of the multiple static images or the inter stimulus interval (ISI) between the images. We found that the size of the face composite effect in the dynamic condition was significantly smaller than that in the static conditions. In other words, the dynamic face display influenced participants to process the target faces in a part-based manner and consequently their recognition of the upper portion of the composite face at test became less interfered with by the aligned lower part of the foil face. The findings from the present experiments provide the strongest evidence to date to suggest that the rigid facial motion mainly influences facial featural, but not holistic, processing. PMID:22342561

Real-time high-resolution measurement of collagen alignment in dynamically loaded soft tissue.

PubMed

York, Timothy; Kahan, Lindsey; Lake, Spencer P; Gruev, Viktor

2014-06-01

A technique for creating maps of the direction and strength of fiber alignment in collagenous soft tissues is presented. The method uses a division of focal plane polarimeter to measure circularly polarized light transmitted through the tissue. The architecture of the sensor allows measurement of the retardance and fiber alignment at the full frame rate of the sensor without any moving optics. The technique compares favorably to the standard method of using a rotating polarizer. How the new technique enables real-time capture of the full angular spread of fiber alignment and retardance under various cyclic loading conditions is illustrated.
Competing mechanisms and scaling laws for carbon nanotube scission by ultrasonication.

PubMed

Pagani, Guido; Green, Micah J; Poulin, Philippe; Pasquali, Matteo

2012-07-17

Dispersion of carbon nanotubes (CNTs) into liquids typically requires ultrasonication to exfoliate individuals CNTs from bundles. Experiments show that CNT length drops with sonication time (or energy) as a power law t(-m). Yet the breakage mechanism is not well understood, and the experimentally reported power law exponent m ranges from approximately 0.2 to 0.5. Here we simulate the motion of CNTs around cavitating bubbles by coupling brownian dynamics with the Rayleigh-Plesset equation. We observe that, during bubble growth, CNTs align tangentially to the bubble surface. Surprisingly, we find two dynamical regimes during the collapse: shorter CNTs align radially, longer ones buckle. We compute the phase diagram for CNT collapse dynamics as a function of CNT length, stiffness, and initial distance from the bubble nuclei and determine the transition from aligning to buckling. We conclude that, depending on their length, CNTs can break due to either buckling or stretching. These two mechanisms yield different power laws for the length decay (0.25 and 0.5, respectively), reconciling the apparent discrepancy in the experimental data.
A Model for Determining Optimal Governance Structure in DoD Acquisition Projects in a Performance-Based Environment

DTIC Science & Technology

2010-04-30

combating market dynamism (Aldrich, 1979; Child, 1972), which is a result of evolving technology, shifting prices, or variance in product availability... principles : (1) human beings are bounded rationally, and (2), as a result of being rationally bound, will always choose to further their own self... principles to govern the relationship among the buyers and suppliers. Our conceptual model aligns the alternative governance structures derived
Aligning Sales Curriculum Content and Pedagogy with Practitioners' Needs

ERIC Educational Resources Information Center

Newberry, Robert; Collins, Marianne K.

2015-01-01

Meeting the instructional needs of both students and sales practitioners is a common challenge for sales educators. The dynamic and ever evolving nature of the sales landscape, in conjunction with the need to align sales curriculum with relevant business practices is the focus of this article. Building on previous research, this study investigates…
Dynamic evaluation of anterior dental alignment in a sample of 8- to 11-year-old children.

PubMed

Lombardo, Luca; Berveglieri, Chiara; Guarneri, Antonio; Siciliani, Giuseppe

2012-06-01

To describe perceptions related to different anterior dental alignments in a sample of 106 children aged between 8- and 11-years-old. We employed dynamic media (videos) showing a smile in four different arrangements (ideal incisal occlusion - N, median diastema - D, incisal crowding - A, protruding incisors - P), with and without general contextual attractiveness. The perception is the same both for the whole face and for the frontal smile alone and there are no significant differences between the answers from male and female interviews. Smiles with normal alignment gain higher scores for esthetics and are associated with more positive qualities. In contrast, smiles with proclined and crowded teeth obtain lower scores. Analysis of the results showed that there are no significant differences between perceptions, sensations and judgments related to smiles presented either as part of the whole face or in only the lower facial third: general facial attractiveness does not influence evaluations of the smile. The study confirms the general tendency to award higher scores to smiles with normal alignment. Copyright © 2012 CEO. Published by Elsevier Masson SAS. All rights reserved.
Matrix metalloproteinases: structures, evolution, and diversification.

PubMed

Massova, I; Kotra, L P; Fridman, R; Mobashery, S

1998-09-01

A comprehensive sequence alignment of 64 members of the family of matrix metalloproteinases (MMPs) for the entire sequences, and subsequently the catalytic and the hemopexin-like domains, have been performed. The 64 MMPs were selected from plants, invertebrates, and vertebrates. The analyses disclosed that as many as 23 distinct subfamilies of these proteins are known to exist. Information from the sequence alignments was correlated with structures, both crystallographic as well as computational, of the catalytic domains for the 23 representative members of the MMP family. A survey of the metal binding sites and two loops containing variable sequences of amino acids, which are important for substrate interactions, are discussed. The collective data support the proposal that the assembly of the domains into multidomain enzymes was likely to be an early evolutionary event. This was followed by diversification, perhaps in parallel among the MMPs, in a subsequent evolutionary time scale. Analysis indicates that a retrograde structure simplification may have accounted for the evolution of MMPs with simple domain constituents, such as matrilysin, from the larger and more elaborate enzymes.
Lepidopteran HMG-CoA reductase is a potential selective target for pest control

PubMed Central

Li, Yuan-mei; Huang, Juan; Tobe, Stephen S.

2017-01-01

As a consequence of the negative impacts on the environment of some insecticides, discovery of eco-friendly insecticides and target has received global attention in recent years. Sequence alignment and structural comparison of the rate-limiting enzyme HMG-CoA reductase (HMGR) revealed differences between lepidopteran pests and other organisms, which suggested insect HMGR could be a selective insecticide target candidate. Inhibition of JH biosynthesis in vitro confirmed that HMGR inhibitors showed a potent lethal effect on the lepidopteran pest Manduca sexta, whereas there was little effect on JH biosynthesis in Apis mellifera and Diploptera punctata. The pest control application of these inhibitors demonstrated that they can be insecticide candidates with potent ovicidal activity, larvicidal activity and insect growth regulatory effects. The present study has validated that Lepidopteran HMGR can be a potent selective insecticide target, and the HMGR inhibitors (especially type II statins) could be selective insecticide candidates and lead compounds. Furthermore, we demonstrated that sequence alignment, homology modeling and structural comparison may be useful for determining potential enzymes or receptors which can be eco-friendly pesticide targets. PMID:28133568
Lepidopteran HMG-CoA reductase is a potential selective target for pest control.

PubMed

Li, Yuan-Mei; Kai, Zhen-Peng; Huang, Juan; Tobe, Stephen S

2017-01-01

As a consequence of the negative impacts on the environment of some insecticides, discovery of eco-friendly insecticides and target has received global attention in recent years. Sequence alignment and structural comparison of the rate-limiting enzyme HMG-CoA reductase (HMGR) revealed differences between lepidopteran pests and other organisms, which suggested insect HMGR could be a selective insecticide target candidate. Inhibition of JH biosynthesis in vitro confirmed that HMGR inhibitors showed a potent lethal effect on the lepidopteran pest Manduca sexta , whereas there was little effect on JH biosynthesis in Apis mellifera and Diploptera punctata . The pest control application of these inhibitors demonstrated that they can be insecticide candidates with potent ovicidal activity, larvicidal activity and insect growth regulatory effects. The present study has validated that Lepidopteran HMGR can be a potent selective insecticide target, and the HMGR inhibitors (especially type II statins) could be selective insecticide candidates and lead compounds. Furthermore, we demonstrated that sequence alignment, homology modeling and structural comparison may be useful for determining potential enzymes or receptors which can be eco-friendly pesticide targets.
Biofuel cells based on direct enzyme-electrode contacts using PQQ-dependent glucose dehydrogenase/bilirubin oxidase and modified carbon nanotube materials.

PubMed

Scherbahn, V; Putze, M T; Dietzel, B; Heinlein, T; Schneider, J J; Lisdat, F

2014-11-15

Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7-1.3 mA/cm(2). The BP-based fuel cell exhibits maximal power density of about 107 µW/cm(2) (at 490 mV). The vaCNT-based fuel cell achieves a maximal power density of 122 µW/cm(2) (at 540 mV). Even after three days and several runs of load a power density over 110 µW/cm(2) is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41 µW/cm(2) (390 mV) can be achieved. Copyright © 2014 Elsevier B.V. All rights reserved.
Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics.

PubMed

Mitchell, Gabriel J; Nelson, Daniel C; Weitz, Joshua S

2010-10-04

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.
Isolation of a cDNA Encoding a Granule-Bound 152-Kilodalton Starch-Branching Enzyme in Wheat1

PubMed Central

Båga, Monica; Nair, Ramesh B.; Repellin, Anne; Scoles, Graham J.; Chibbar, Ravindra N.

2000-01-01

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5′-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80–100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum. PMID:10982440
What can the past of pay-for-performance tell us about the future of Value-Based Purchasing in Medicare?

PubMed

Ryan, Andrew M; Damberg, Cheryl L

2013-06-01

The Medicare program has implemented pay-for-performance (P4P), or Value-Based Purchasing, for inpatient care and for Medicare Advantage plans, and plans to implement a program for physicians in 2015. In this paper, we review evidence on the effectiveness of P4P and identify design criteria deemed to be best practice in P4P. We then assess the extent to which Medicare's existing and planned Value-Based Purchasing programs align with these best practices. Of the seven identified best practices in P4P program design, the Hospital Value-Based Purchasing program is strongly aligned with two of the best practices, moderately aligned with three, weakly aligned with one, and has unclear alignment with one best practice. The Physician Value-Based Purchasing Modifier is strongly aligned with two of the best practices, moderately aligned with one, weakly aligned with three, and has unclear alignment with one of the best practices. The Medicare Advantage Quality Bonus Program is strongly aligned with four of the best practices, moderately aligned with two, and weakly aligned with one of the best practices. We identify enduring gaps in P4P literature as it relates to Medicare's plans for Value-Based Purchasing and discuss important issues in the future of these implementations in Medicare. Copyright © 2013 Elsevier Inc. All rights reserved.
Frontiers of controlling energy levels at interfaces

NASA Astrophysics Data System (ADS)

Koch, Norbert

The alignment of electron energy levels at interfaces between semiconductors, dielectrics, and electrodes determines the function and efficiency of all electronic and optoelectronic devices. Reliable guidelines for predicting the level alignment for a given material combination and methods to adjust the intrinsic energy landscape are needed to enable efficient engineering approaches. These are sufficiently understood for established electronic materials, e.g., Si, but for the increasing number of emerging materials, e.g., organic and 2D semiconductors, perovskites, this is work in progress. The intrinsic level alignment and the underlying mechanisms at interfaces between organic and inorganic semiconductors are discussed first. Next, methods to alter the level alignment are introduced, which all base on proper charge density rearrangement at a heterojunction. As interface modification agents we use molecular electron acceptors and donors, as well as molecular photochromic switches that add a dynamic aspect and allow device multifunctionality. For 2D semiconductors surface transfer doping with molecular acceptors/donors transpires as viable method to locally tune the Fermi-level position in the energy gap. The fundamental electronic properties of a prototypical 1D interface between intrinsic and p-doped 2D semiconductor regions are derived from local (scanning probe) and area-averaged (photoemission) spectroscopy experiments. Future research opportunities for attaining unsurpassed interface control through charge density management are discussed.
Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926
Transition-State Ensembles Navigate the Pathways of Enzyme Catalysis.

PubMed

Mickert, Matthias J; Gorris, Hans H

2018-06-07

Transition-state theory (TST) provides an important framework for analyzing and explaining the reaction rates of enzymes. TST, however, needs to account for protein dynamic effects and heterogeneities in enzyme catalysis. We have analyzed the reaction rates of β-galactosidase and β-glucuronidase at the single molecule level by using large arrays of femtoliter-sized chambers. Heterogeneities in individual reaction rates yield information on the intrinsic distribution of the free energy of activation (Δ G ‡ ) in an enzyme ensemble. The broader distribution of Δ G ‡ in β-galactosidase compared to β-glucuronidase is attributed to β-galactosidase's multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of β-galactosidase, we show that transition-state ensembles do not only contribute to enzyme catalysis but can also channel the catalytic pathway to the formation of different products. We conclude that β-galactosidase is an example of natural evolution, where a new catalytic pathway branches off from an established enzyme function. The functional division of work between enzymatic substates explains why the conformational space represented by the enzyme ensemble is larger than the conformational space that can be sampled by any given enzyme molecule during catalysis.
Effects of flow on the dynamics of a ferromagnetic nematic liquid crystal

NASA Astrophysics Data System (ADS)

Potisk, Tilen; Pleiner, Harald; Svenšek, Daniel; Brand, Helmut R.

2018-04-01

We investigate the effects of flow on the dynamics of ferromagnetic nematic liquid crystals. As a model, we study the coupled dynamics of the magnetization, M , the director field, n , associated with the liquid crystalline orientational order, and the velocity field, v . We evaluate how simple shear flow in a ferromagnetic nematic is modified in the presence of small external magnetic fields, and we make experimentally testable predictions for the resulting effective shear viscosity: an increase by a factor of 2 in a magnetic field of about 20 mT. Flow alignment, a characteristic feature of classical uniaxial nematic liquid crystals, is analyzed for ferromagnetic nematics for the two cases of magnetization in or perpendicular to the shear plane. In the former case, we find that small in-plane magnetic fields are sufficient to suppress tumbling and thus that the boundary between flow alignment and tumbling can be controlled easily. In the latter case, we furthermore find a possibility of flow alignment in a regime for which one obtains tumbling for the pure nematic component. We derive the analogs of the three Miesowicz viscosities well-known from usual nematic liquid crystals, corresponding to nine different configurations. Combinations of these can be used to determine several dynamic coefficients experimentally.
Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides

DOE PAGES

Ramalho, Teodorico C.; DeCastro, Alexandre A.; Silva, Daniela R.; ...

2015-08-26

The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and helpmore » in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.« less
Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides

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

Ramalho, Teodorico C.; DeCastro, Alexandre A.; Silva, Daniela R.

The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and helpmore » in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.« less
Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition.

PubMed

Huang, Yuhong; Busk, Peter Kamp; Grell, Morten Nedergaard; Zhao, Hai; Lange, Lene

2014-12-01

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation. Copyright © 2014 Elsevier Inc. All rights reserved.
The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes.

PubMed

Treangen, Todd J; Ondov, Brian D; Koren, Sergey; Phillippy, Adam M

2014-01-01

Whole-genome sequences are now available for many microbial species and clades, however existing whole-genome alignment methods are limited in their ability to perform sequence comparisons of multiple sequences simultaneously. Here we present the Harvest suite of core-genome alignment and visualization tools for the rapid and simultaneous analysis of thousands of intraspecific microbial strains. Harvest includes Parsnp, a fast core-genome multi-aligner, and Gingr, a dynamic visual platform. Together they provide interactive core-genome alignments, variant calls, recombination detection, and phylogenetic trees. Using simulated and real data we demonstrate that our approach exhibits unrivaled speed while maintaining the accuracy of existing methods. The Harvest suite is open-source and freely available from: http://github.com/marbl/harvest.

Classification of biosensor time series using dynamic time warping: applications in screening cancer cells with characteristic biomarkers.

PubMed

Rai, Shesh N; Trainor, Patrick J; Khosravi, Farhad; Kloecker, Goetz; Panchapakesan, Balaji

2016-01-01

The development of biosensors that produce time series data will facilitate improvements in biomedical diagnostics and in personalized medicine. The time series produced by these devices often contains characteristic features arising from biochemical interactions between the sample and the sensor. To use such characteristic features for determining sample class, similarity-based classifiers can be utilized. However, the construction of such classifiers is complicated by the variability in the time domains of such series that renders the traditional distance metrics such as Euclidean distance ineffective in distinguishing between biological variance and time domain variance. The dynamic time warping (DTW) algorithm is a sequence alignment algorithm that can be used to align two or more series to facilitate quantifying similarity. In this article, we evaluated the performance of DTW distance-based similarity classifiers for classifying time series that mimics electrical signals produced by nanotube biosensors. Simulation studies demonstrated the positive performance of such classifiers in discriminating between time series containing characteristic features that are obscured by noise in the intensity and time domains. We then applied a DTW distance-based k -nearest neighbors classifier to distinguish the presence/absence of mesenchymal biomarker in cancer cells in buffy coats in a blinded test. Using a train-test approach, we find that the classifier had high sensitivity (90.9%) and specificity (81.8%) in differentiating between EpCAM-positive MCF7 cells spiked in buffy coats and those in plain buffy coats.
Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

PubMed Central

Rehberg, Markus; Ritter, Joachim B.; Reichl, Udo

2014-01-01

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model's predictive power supports the design of more efficient bioprocesses. PMID:25329309
Glycolysis is governed by growth regime and simple enzyme regulation in adherent MDCK cells.

PubMed

Rehberg, Markus; Ritter, Joachim B; Reichl, Udo

2014-10-01

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model's predictive power supports the design of more efficient bioprocesses.
Coupled chemical reactions in dynamic nanometric confinement: VII. Biosensors based on swift heavy ion tracks with membranes

NASA Astrophysics Data System (ADS)

Fink, D.; Muñoz H., G.; Garcia-Arrelano, H.; Alfonta, L.; Vacik, J.; Kiv, A.; Hnatowicz, V.

2017-02-01

In previous papers it was shown that the coupling of the two chemical reactions: {NaOH etchant - PET polymer} and {NaOH etchant - AgNO3 solution} within the dynamic confinement of etched swift heavy ion tracks eventually leads to the formation of tiny Ag2O membranes within these nanopores, thus separating the latter ones into two adjacent segments. It is shown here that the deposition of enzymes in these two segments transforms these structures into biosensors. In our earlier developed sensors with transparent etched ion tracks, we frequently used glucose oxidase as enzyme and glucose as analyte. In these cases, the enzymatic reaction within the tracks leads to a change in the pH value of the confined solution and hence also in the track conductivity, so these structures can be used for biosensing. When applying, for easy comparison, the same enzyme/analyte combination to the segmented sensor arrangement presented here, we find a striking improvement in detection sensitivity which points at a different biosensing mechanism due to intrinsic polarisation effects across the newly inserted membranes.
In-silico studies of neutral drift for functional protein interaction networks

NASA Astrophysics Data System (ADS)

Ali, Md Zulfikar; Wingreen, Ned S.; Mukhopadhyay, Ranjan

We have developed a minimal physically-motivated model of protein-protein interaction networks. Our system consists of two classes of enzymes, activators (e.g. kinases) and deactivators (e.g. phosphatases), and the enzyme-mediated activation/deactivation rates are determined by sequence-dependent binding strengths between enzymes and their targets. The network is evolved by introducing random point mutations in the binding sequences where we assume that each new mutation is either fixed or entirely lost. We apply this model to studies of neutral drift in networks that yield oscillatory dynamics, where we start, for example, with a relatively simple network and allow it to evolve by adding nodes and connections while requiring that dynamics be conserved. Our studies demonstrate both the importance of employing a sequence-based evolutionary scheme and the relative rapidity (in evolutionary time) for the redistribution of function over new nodes via neutral drift. Surprisingly, in addition to this redistribution time we discovered another much slower timescale for network evolution, reflecting hidden order in sequence space that we interpret in terms of sparsely connected domains.
Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.
Evaluation of an image-based tracking workflow with Kalman filtering for automatic image plane alignment in interventional MRI.

PubMed

Neumann, M; Cuvillon, L; Breton, E; de Matheli, M

2013-01-01

Recently, a workflow for magnetic resonance (MR) image plane alignment based on tracking in real-time MR images was introduced. The workflow is based on a tracking device composed of 2 resonant micro-coils and a passive marker, and allows for tracking of the passive marker in clinical real-time images and automatic (re-)initialization using the microcoils. As the Kalman filter has proven its benefit as an estimator and predictor, it is well suited for use in tracking applications. In this paper, a Kalman filter is integrated in the previously developed workflow in order to predict position and orientation of the tracking device. Measurement noise covariances of the Kalman filter are dynamically changed in order to take into account that, according to the image plane orientation, only a subset of the 3D pose components is available. The improved tracking performance of the Kalman extended workflow could be quantified in simulation results. Also, a first experiment in the MRI scanner was performed but without quantitative results yet.
Evaluation of the interaction of cyclin-dependent kinase 5 with activator p25 and with p25-derived inhibitor CIP.

PubMed

Cardone, Antonio; Albers, R Wayne; Sriram, Ram D; Pant, Harish C

2010-05-01

A high-affinity inhibitor protein called CIP, produced by small truncations of p35, was experimentally identified. P35 is a physiological activator of the cyclin-dependent kinase cdk5. P25 is derived from proteolytic truncation of p35 within "stressed" neurons, and it is associated with the hyperphosphorylation of specific neuronal proteins, typically occurring in neurodegenerative diseases such as Alzheimer's. Here, we report a study of the binding mechanisms of the cdk5-p25 and cdk5-CIP complexes. This provides a better understanding of the source of the inhibitory activity of the protein CIP. We use a geometry-based technique to test the hypothesis that p25's truncation increases the flexibility of CIP and thus prevents cdk5 from reaching its active conformation. Our study is based on a geometry-based alignment algorithm, which aligns two given protein conformations with respect to their interfaces. Our results support the flexibility hypothesis and will be used as a basis for targeted molecular dynamics simulations.
Automated protein structure modeling in CASP9 by I-TASSER pipeline combined with QUARK-based ab initio folding and FG-MD-based structure refinement

PubMed Central

Xu, Dong; Zhang, Jian; Roy, Ambrish; Zhang, Yang

2011-01-01

I-TASSER is an automated pipeline for protein tertiary structure prediction using multiple threading alignments and iterative structure assembly simulations. In CASP9 experiments, two new algorithms, QUARK and FG-MD, were added to the I-TASSER pipeline for improving the structural modeling accuracy. QUARK is a de novo structure prediction algorithm used for structure modeling of proteins that lack detectable template structures. For distantly homologous targets, QUARK models are found useful as a reference structure for selecting good threading alignments and guiding the I-TASSER structure assembly simulations. FG-MD is an atomic-level structural refinement program that uses structural fragments collected from the PDB structures to guide molecular dynamics simulation and improve the local structure of predicted model, including hydrogen-bonding networks, torsion angles and steric clashes. Despite considerable progress in both the template-based and template-free structure modeling, significant improvements on protein target classification, domain parsing, model selection, and ab initio folding of beta-proteins are still needed to further improve the I-TASSER pipeline. PMID:22069036
Energy absorption ability of buckyball C720 at low impact speed: a numerical study based on molecular dynamics

PubMed Central

2013-01-01

The dynamic impact response of giant buckyball C720 is investigated by using molecular dynamics simulations. The non-recoverable deformation of C720 makes it an ideal candidate for high-performance energy absorption. Firstly, mechanical behaviors under dynamic impact and low-speed crushing are simulated and modeled, which clarifies the buckling-related energy absorption mechanism. One-dimensional C720 arrays (both vertical and horizontal alignments) are studied at various impact speeds, which show that the energy absorption ability is dominated by the impact energy per buckyball and less sensitive to the number and arrangement direction of buckyballs. Three-dimensional stacking of buckyballs in simple cubic, body-centered cubic, hexagonal, and face-centered cubic forms are investigated. Stacking form with higher occupation density yields higher energy absorption. The present study may shed lights on employing C720 assembly as an advanced energy absorption system against low-speed impacts. PMID:23360618
Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications

PubMed Central

2014-01-01

Background Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. Results A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. Conclusions The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications. PMID:24485248
Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications.

PubMed

Li, Chunfang; Du, Miaofen; Cheng, Bin; Wang, Lushan; Liu, Xinqiang; Ma, Cuiqing; Yang, Chunyu; Xu, Ping

2014-01-31

Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.
Protein Structure and Function Prediction Using I-TASSER

PubMed Central

Yang, Jianyi; Zhang, Yang

2016-01-01

I-TASSER is a hierarchical protocol for automated protein structure prediction and structure-based function annotation. Starting from the amino acid sequence of target proteins, I-TASSER first generates full-length atomic structural models from multiple threading alignments and iterative structural assembly simulations followed by atomic-level structure refinement. The biological functions of the protein, including ligand-binding sites, enzyme commission number, and gene ontology terms, are then inferred from known protein function databases based on sequence and structure profile comparisons. I-TASSER is freely available as both an on-line server and a stand-alone package. This unit describes how to use the I-TASSER protocol to generate structure and function prediction and how to interpret the prediction results, as well as alternative approaches for further improving the I-TASSER modeling quality for distant-homologous and multi-domain protein targets. PMID:26678386
Orbit Alignment in Triple Stars

NASA Astrophysics Data System (ADS)

Tokovinin, Andrei

2017-08-01

The statistics of the angle Φ between orbital angular momenta in hierarchical triple systems with known inner visual or astrometric orbits are studied. A correlation between apparent revolution directions proves the partial orbit alignment known from earlier works. The alignment is strong in triples with outer projected separation less than ˜50 au, where the average Φ is about 20^\\circ . In contrast, outer orbits wider than 1000 au are not aligned with the inner orbits. It is established that the orbit alignment decreases with the increasing mass of the primary component. The average eccentricity of inner orbits in well-aligned triples is smaller than in randomly aligned ones. These findings highlight the role of dissipative interactions with gas in defining the orbital architecture of low-mass triple systems. On the other hand, chaotic dynamics apparently played a role in shaping more massive hierarchies. The analysis of projected configurations and triples with known inner and outer orbits indicates that the distribution of Φ is likely bimodal, where 80% of triples have {{Φ }}< 70^\\circ and the remaining ones are randomly aligned.
Identification and Analysis of Jasmonate Pathway Genes in Coffea canephora (Robusta Coffee) by In Silico Approach.

PubMed

Bharathi, Kosaraju; Sreenath, H L

2017-07-01

Coffea canephora is the commonly cultivated coffee species in the world along with Coffea arabica . Different pests and pathogens affect the production and quality of the coffee. Jasmonic acid (JA) is a plant hormone which plays an important role in plants growth, development, and defense mechanisms, particularly against insect pests. The key enzymes involved in the production of JA are lipoxygenase, allene oxide synthase, allene oxide cyclase, and 12-oxo-phytodienoic reductase. There is no report on the genes involved in JA pathway in coffee plants. We made an attempt to identify and analyze the genes coding for these enzymes in C. canephora . First, protein sequences of jasmonate pathway genes from model plant Arabidopsis thaliana were identified in the National Center for Biotechnology Information (NCBI) database. These protein sequences were used to search the web-based database Coffee Genome Hub to identify homologous protein sequences in C. canephora genome using Basic Local Alignment Search Tool (BLAST). Homologous protein sequences for key genes were identified in the C. canephora genome database. Protein sequences of the top matches were in turn used to search in NCBI database using BLAST tool to confirm the identity of the selected proteins and to identify closely related genes in species. The protein sequences from C. canephora database and the top matches in NCBI were aligned, and phylogenetic trees were constructed using MEGA6 software and identified the genetic distance of the respective genes. The study identified the four key genes of JA pathway in C. canephora , confirming the conserved nature of the pathway in coffee. The study expected to be useful to further explore the defense mechanisms of coffee plants. JA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC, and OPR are identified in C. canephora (robusta coffee) by bioinformatic approaches confirming the conserved nature of the pathway in coffee. The findings are useful to understand the defense mechanisms of C. canephora and coffee breeding in the long run. JA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC and OPR were identified and analyzed in C. canephora (robusta coffee) by in silico approach. The study has confirmed the conserved nature of JA pathway in coffee; the findings are useful to further explore the defense mechanisms of coffee plants. Abbreviations used: C. canephora : Coffea canephora ; C. arabica : Coffea arabica ; JA: Jasmonic acid; CGH: Coffee Genome Hub; NCBI: National Centre for Biotechnology Information; BLAST: Basic Local Alignment Search Tool; A. thaliana : Arabidopsis thaliana ; LOX: Lipoxygenase, AOS: Allene oxide synthase; AOC: Allene oxide cyclase; OPR: 12 oxo phytodienoic reductase.
Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

NASA Astrophysics Data System (ADS)

Dodani, Sheel C.; Kiss, Gert; Cahn, Jackson K. B.; Su, Ye; Pande, Vijay S.; Arnold, Frances H.

2016-05-01

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.
Slider--maximum use of probability information for alignment of short sequence reads and SNP detection.

PubMed

Malhis, Nawar; Butterfield, Yaron S N; Ester, Martin; Jones, Steven J M

2009-01-01

A plethora of alignment tools have been created that are designed to best fit different types of alignment conditions. While some of these are made for aligning Illumina Sequence Analyzer reads, none of these are fully utilizing its probability (prb) output. In this article, we will introduce a new alignment approach (Slider) that reduces the alignment problem space by utilizing each read base's probabilities given in the prb files. Compared with other aligners, Slider has higher alignment accuracy and efficiency. In addition, given that Slider matches bases with probabilities other than the most probable, it significantly reduces the percentage of base mismatches. The result is that its SNP predictions are more accurate than other SNP prediction approaches used today that start from the most probable sequence, including those using base quality.
Irreversible covalent modification of type I dehydroquinase with a stable Schiff base.

PubMed

Tizón, Lorena; Maneiro, María; Peón, Antonio; Otero, José M; Lence, Emilio; Poza, Sergio; van Raaij, Mark J; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

2015-01-21

The irreversible inhibition of type I dehydroquinase (DHQ1), the third enzyme of the shikimic acid pathway, is investigated by structural, biochemical and computational studies. Two epoxides, which are mimetics of the natural substrate, were designed as irreversible inhibitors of the DHQ1 enzyme and to study the binding requirements of the linkage to the enzyme. The epoxide with the S configuration caused the covalent modification of the protein whereas no reaction was obtained with its epimer. The first crystal structure of DHQ1 from Salmonella typhi covalently modified by the S epoxide, which is reported at 1.4 Å, revealed that the modified ligand is surprisingly covalently attached to the essential Lys170 by the formation of a stable Schiff base. The experimental and molecular dynamics simulation studies reported here highlight the huge importance of the conformation of the C3 carbon of the ligand for covalent linkage to this type of aldolase I enzyme, revealed the key role played by the essential His143 as a Lewis acid in this process and show the need for a neatly closed active site for catalysis.
Synthesis of pH-responsive β-CD-based star polymer and impact of its self-assembly behavior on pectinase activity.

PubMed

Hu, Dong; Yang, Hong; Liu, Jiangtao; Lei, Zhongli

2017-03-01

A novel type of pH-responsive star polymer based on β-cyclodextrin (β-CD) was synthesized and further covalently conjugated with enzyme. The impact of its self-assembly behavior on enzyme activity was investigated. In our design, azide containing the polymer (N 3 ) 7 -β-CD-(PtBA) 14 was synthesized via atom transfer radical polymerization of tert-butyl acrylate using (N 3 ) 7 -β-CD-(Br) 14 as the multifunctional initiator. The final product (N 3 ) 7 -β-CD-(PAA) 14 was obtained via hydrolysis and covalently conjugating pectinase onto pH-responsive polyacrylic acid (PAA) arms. PAA can change its conformation with the self-assembly by altered pH, leading its nanostructure into micellar nanoparticles in aqueous solution and further affecting the activity of immobilized pectinase. The results were proved by fluorescence spectroscopy and dynamic light scattering. This system proves that the activity of immobilized enzyme can be tailored predictably, and this pH-responsive polymer holds great potential for controllable delivery of enzymes. © 2016 International Union of Biochemistry and Molecular Biology, Inc.
Effect of Alignment on L2 Written Production

ERIC Educational Resources Information Center

Wang, Chuming; Wang, Min

2015-01-01

This article aims to uncover how L2 writing is affected by alignment, a socio-cognitive process involving dynamic coordination and adaptation. For this, two studies were conducted. Study 1 required two groups of 24 learners of English as a foreign language (EFL) to continue in English two stories with their endings removed, both of which had a…

Prediction of enzyme classes from 3D structure: a general model and examples of experimental-theoretic scoring of peptide mass fingerprints of Leishmania proteins.

PubMed

Concu, Riccardo; Dea-Ayuela, Maria A; Perez-Montoto, Lazaro G; Bolas-Fernández, Francisco; Prado-Prado, Francisco J; Podda, Gianni; Uriarte, Eugenio; Ubeira, Florencio M; González-Díaz, Humberto

2009-09-01

The number of protein and peptide structures included in Protein Data Bank (PDB) and Gen Bank without functional annotation has increased. Consequently, there is a high demand for theoretical models to predict these functions. Here, we trained and validated, with an external set, a Markov Chain Model (MCM) that classifies proteins by their possible mechanism of action according to Enzyme Classification (EC) number. The methodology proposed is essentially new, and enables prediction of all EC classes with a single equation without the need for an equation for each class or nonlinear models with multiple outputs. In addition, the model may be used to predict whether one peptide presents a positive or negative contribution of the activity of the same EC class. The model predicts the first EC number for 106 out of 151 (70.2%) oxidoreductases, 178/178 (100%) transferases, 223/223 (100%) hydrolases, 64/85 (75.3%) lyases, 74/74 (100%) isomerases, and 100/100 (100%) ligases, as well as 745/811 (91.9%) nonenzymes. It is important to underline that this method may help us predict new enzyme proteins or select peptide candidates that improve enzyme activity, which may be of interest for the prediction of new drugs or drug targets. To illustrate the model's application, we report the 2D-Electrophoresis (2DE) isolation from Leishmania infantum as well as MADLI TOF Mass Spectra characterization and theoretical study of the Peptide Mass Fingerprints (PMFs) of a new protein sequence. The theoretical study focused on MASCOT, BLAST alignment, and alignment-free QSAR prediction of the contribution of 29 peptides found in the PMF of the new protein to specific enzyme action. This combined strategy may be used to identify and predict peptides of prokaryote and eukaryote parasites and their hosts as well as other superior organisms, which may be of interest in drug development or target identification.
Dynamic Structure-Based Pharmacophore Model Development: A New and Effective Addition in the Histone Deacetylase 8 (HDAC8) Inhibitor Discovery

PubMed Central

Thangapandian, Sundarapandian; John, Shalini; Lee, Yuno; Kim, Songmi; Lee, Keun Woo

2011-01-01

Histone deacetylase 8 (HDAC8) is an enzyme involved in deacetylating the amino groups of terminal lysine residues, thereby repressing the transcription of various genes including tumor suppressor gene. The over expression of HDAC8 was observed in many cancers and thus inhibition of this enzyme has emerged as an efficient cancer therapeutic strategy. In an effort to facilitate the future discovery of HDAC8 inhibitors, we developed two pharmacophore models containing six and five pharmacophoric features, respectively, using the representative structures from two molecular dynamic (MD) simulations performed in Gromacs 4.0.5 package. Various analyses of trajectories obtained from MD simulations have displayed the changes upon inhibitor binding. Thus utilization of the dynamically-responded protein structures in pharmacophore development has the added advantage of considering the conformational flexibility of protein. The MD trajectories were clustered based on single-linkage method and representative structures were taken to be used in the pharmacophore model development. Active site complimenting structure-based pharmacophore models were developed using Discovery Studio 2.5 program and validated using a dataset of known HDAC8 inhibitors. Virtual screening of chemical database coupled with drug-like filter has identified drug-like hit compounds that match the pharmacophore models. Molecular docking of these hits reduced the false positives and identified two potential compounds to be used in future HDAC8 inhibitor design. PMID:22272142
Second Language Developmental Dynamics: How Dynamic Systems Theory Accounts for Issues in Second Language Learning

ERIC Educational Resources Information Center

Rosmawati

2014-01-01

Dynamic systems theory (DST) is presented in this article as a suitable approach to research the acquisition of second language (L2) because of its close alignment with the process of second language learning. Through a process of identifying and comparing the characteristics of a dynamic system with the process of L2 learning, this article…
Alignment and integration of complex networks by hypergraph-based spectral clustering

NASA Astrophysics Data System (ADS)

Michoel, Tom; Nachtergaele, Bruno

2012-11-01

Complex networks possess a rich, multiscale structure reflecting the dynamical and functional organization of the systems they model. Often there is a need to analyze multiple networks simultaneously, to model a system by more than one type of interaction, or to go beyond simple pairwise interactions, but currently there is a lack of theoretical and computational methods to address these problems. Here we introduce a framework for clustering and community detection in such systems using hypergraph representations. Our main result is a generalization of the Perron-Frobenius theorem from which we derive spectral clustering algorithms for directed and undirected hypergraphs. We illustrate our approach with applications for local and global alignment of protein-protein interaction networks between multiple species, for tripartite community detection in folksonomies, and for detecting clusters of overlapping regulatory pathways in directed networks.
Alignment and integration of complex networks by hypergraph-based spectral clustering.

PubMed

Michoel, Tom; Nachtergaele, Bruno

2012-11-01

Complex networks possess a rich, multiscale structure reflecting the dynamical and functional organization of the systems they model. Often there is a need to analyze multiple networks simultaneously, to model a system by more than one type of interaction, or to go beyond simple pairwise interactions, but currently there is a lack of theoretical and computational methods to address these problems. Here we introduce a framework for clustering and community detection in such systems using hypergraph representations. Our main result is a generalization of the Perron-Frobenius theorem from which we derive spectral clustering algorithms for directed and undirected hypergraphs. We illustrate our approach with applications for local and global alignment of protein-protein interaction networks between multiple species, for tripartite community detection in folksonomies, and for detecting clusters of overlapping regulatory pathways in directed networks.
Alignment of cell division axes in directed epithelial cell migration

NASA Astrophysics Data System (ADS)

Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

2014-11-01

Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.
WEB-server for search of a periodicity in amino acid and nucleotide sequences

NASA Astrophysics Data System (ADS)

E Frenkel, F.; Skryabin, K. G.; Korotkov, E. V.

2017-12-01

A new web server (http://victoria.biengi.ac.ru/splinter/login.php) was designed and developed to search for periodicity in nucleotide and amino acid sequences. The web server operation is based upon a new mathematical method of searching for multiple alignments, which is founded on the position weight matrices optimization, as well as on implementation of the two-dimensional dynamic programming. This approach allows the construction of multiple alignments of the indistinctly similar amino acid and nucleotide sequences that accumulated more than 1.5 substitutions per a single amino acid or a nucleotide without performing the sequences paired comparisons. The article examines the principles of the web server operation and two examples of studying amino acid and nucleotide sequences, as well as information that could be obtained using the web server.
Electric fields and field-aligned currents in polar regions of the solar corona: 3-D MHD consideration

NASA Technical Reports Server (NTRS)

Pisanko, Yu. V.

1995-01-01

The calculation of the solar rotation electro-dynamical effects in the near-the-Sun solar wind seems more convenient from the non-inertial corotating reference frame. This implies some modification of the 3-D MHD equations generally on the base of the General Theory of Relativity. The paper deals with the search of stationary (in corotating non-inertial reference frame) solutions of the modified 3-D MHD equations for the in near-the-Sun high latitude sub-alfvenic solar wind. The solution is obtained requiring electric fields and field-aligned electric currents in the high latitude near-the-Sun solar wind. Various scenario are explored self-consistently via a number of numerical experiments. The analogy with the high latitude Earth's magnetosphere is used for the interpretation of the results. Possible observational manifestations are discussed.
TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics

PubMed Central

Röst, Hannes L.; Liu, Yansheng; D’Agostino, Giuseppe; Zanella, Matteo; Navarro, Pedro; Rosenberger, George; Collins, Ben C.; Gillet, Ludovic; Testa, Giuseppe; Malmström, Lars; Aebersold, Ruedi

2016-01-01

Large scale, quantitative proteomic studies have become essential for the analysis of clinical cohorts, large perturbation experiments and systems biology studies. While next-generation mass spectrometric techniques such as SWATH-MS have substantially increased throughput and reproducibility, ensuring consistent quantification of thousands of peptide analytes across multiple LC-MS/MS runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion, we have developed the TRIC software which utilizes fragment ion data to perform cross-run alignment, consistent peak-picking and quantification for high throughput targeted proteomics. TRIC uses a graph-based alignment strategy based on non-linear retention time correction to integrate peak elution information from all LC-MS/MS runs acquired in a study. When compared to state-of-the-art SWATH-MS data analysis, the algorithm was able to reduce the identification error by more than 3-fold at constant recall, while correcting for highly non-linear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem (iPS) cells, TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups and substantially increased the quantitative completeness and biological information in the data, providing insights into protein dynamics of iPS cells. Overall, this study demonstrates the importance of consistent quantification in highly challenging experimental setups, and proposes an algorithm to automate this task, constituting the last missing piece in a pipeline for automated analysis of massively parallel targeted proteomics datasets. PMID:27479329
On-command on/off switching of progenitor cell and cancer cell polarized motility and aligned morphology via a cytocompatible shape memory polymer scaffold.

PubMed

Wang, Jing; Quach, Andy; Brasch, Megan E; Turner, Christopher E; Henderson, James H

2017-09-01

In vitro biomaterial models have enabled advances in understanding the role of extracellular matrix (ECM) architecture in the control of cell motility and polarity. Most models are, however, static and cannot mimic dynamic aspects of in vivo ECM remodeling and function. To address this limitation, we present an electrospun shape memory polymer scaffold that can change fiber alignment on command under cytocompatible conditions. Cellular response was studied using the human fibrosarcoma cell line HT-1080 and the murine mesenchymal stem cell line C3H/10T1/2. The results demonstrate successful on-command on/off switching of cell polarized motility and alignment. Decrease in fiber alignment causes a change from polarized motility along the direction of fiber alignment to non-polarized motility and from aligned to unaligned morphology, while increase in fiber alignment causes a change from non-polarized to polarized motility along the direction of fiber alignment and from unaligned to aligned morphology. In addition, the findings are consistent with the hypothesis that increased fiber alignment causes increased cell velocity, while decreased fiber alignment causes decreased cell velocity. On-command on/off switching of cell polarized motility and alignment is anticipated to enable new study of directed cell motility in tumor metastasis, in cell homing, and in tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.
On aggregation in CA models in biology

NASA Astrophysics Data System (ADS)

Alber, Mark S.; Kiskowski, Audi

2001-12-01

Aggregation of randomly distributed particles into clusters of aligned particles is modeled using a cellular automata (CA) approach. The CA model accounts for interactions between more than one type of particle, in which pressures for angular alignment with neighbors compete with pressures for grouping by cell type. In the case of only one particle type clusters tend to unite into one big cluster. In the case of several types of particles the dynamics of clusters is more complicated and for specific choices of parameters particle sorting occurs simultaneously with the formation of clusters of aligned particles.
Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.
Homologues of insulinase, a new superfamily of metalloendopeptidases.

PubMed Central

Rawlings, N D; Barrett, A J

1991-01-01

On the basis of a statistical analysis of an alignment of the amino acid sequences, a new superfamily of metalloendopeptidases is proposed, consisting of human insulinase, Escherichia coli protease III and mitochondrial processing endopeptidases from Saccharomyces and Neurospora. These enzymes do not contain the 'HEXXH' consensus sequence found in all previously recognized zinc metalloendopeptidases. PMID:2025223
Vacuum selection on axionic landscapes

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

Wang, Gaoyuan; Battefeld, Thorsten, E-mail: gaoyuan.wang@stud.uni-goettingen.de, E-mail: tbattefe@astro.physik.uni-goettingen.de

2016-04-01

We compute the distribution of minima that are reached dynamically on multi-field axionic landscapes, both numerically and analytically. Such landscapes are well suited for inflationary model building due to the presence of shift symmetries and possible alignment effects (the KNP mechanism). The resulting distribution of dynamically reached minima differs considerably from the naive expectation based on counting all vacua. These differences are more pronounced in the presence of many fields due to dynamical selection effects: while low lying minima are preferred as fields roll down the potential, trajectories are also more likely to get trapped by one of the manymore » nearby minima. We show that common analytic arguments based on random matrix theory in the large D-limit to estimate the distribution of minima are insufficient for quantitative arguments pertaining to the dynamically reached ones. This discrepancy is not restricted to axionic potentials. We provide an empirical expression for the expectation value of such dynamically reached minimas' height and argue that the cosmological constant problem is not alleviated in the absence of anthropic arguments. We further comment on the likelihood of inflation on axionic landscapes in the large D-limit.« less
Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP.

PubMed

Bickford, Justin S; Nick, Harry S

2013-12-01

Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substrate-specific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.
Transcriptome identification of putative genes involved in protein catabolism and innate immune response in human body louse (Pediculicidae: Pediculus humanus).

PubMed

Pedra, Joao H F; Brandt, Amanda; Li, Hong-Mei; Westerman, Rick; Romero-Severson, Jeanne; Pollack, Richard J; Murdock, Larry L; Pittendrigh, Barry R

2003-11-01

Genomics information relating to human body lice is surprisingly scarce, and this has constrained studies of their physiology, immunology and vector biology. To identify novel body louse genes, we used engorged adult lice to generate a cDNA library. Initially, 1152 clones were screened for inserts, edited for removal of vector sequences and base pairs of poor quality, and viewed for splicing variations, gene families and polymorphism. Computational methods identified 506 inferred open reading frames including the first predicted louse defensin. The inferred defensin aligns well with other insect defensins and has highly conserved cysteine residues, as are known for other defensin sequences. Two cysteine and five serine proteinases were categorized according to their inferred catalytic sites. We also discovered seven putative ubiquitin-pathway genes and four iron metabolizing deduced enzymes. Finally, glutathione-S-transferases and cytochrome P450 genes were among the detoxification enzymes found. Results from this first systematic effort to discover human body louse genes should promote further studies in Phthiraptera and lice.
Modification of S-Adenosyl-l-Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation

PubMed Central

Tambunan, Usman Sumo Friend; Nasution, Mochammad Arfin Fardiansyah; Azhima, Fauziah; Parikesit, Arli Aditya; Toepak, Erwin Prasetya; Idrus, Syarifuddin; Kerami, Djati

2017-01-01

Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. Nonstructural protein 5 (NS5) methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S-adenosyl-l-methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2′OH, resulting in S-adenosyl-l-homocysteine (SAH). The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) test. The 2 simulations were performed using Molecular Operating Environment (MOE) 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356) based on ΔGbinding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever. PMID:28469408
Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

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

Agarwal, Pratul K

2012-01-01

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted thatmore » mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.« less
Are there dynamical effects in enzyme catalysis? Some thoughts concerning the enzymatic chemical step.

PubMed

Tuñón, Iñaki; Laage, Damien; Hynes, James T

2015-09-15

We offer some thoughts on the much debated issue of dynamical effects in enzyme catalysis, and more specifically on their potential role in the acceleration of the chemical step. Since the term 'dynamics' has been used with different meanings, we find it useful to first return to the Transition State Theory rate constant, its assumptions and the choices it involves, and detail the various sources of deviations from it due to dynamics (or not). We suggest that much can be learned about the key current questions for enzyme catalysis from prior extensive studies of dynamical and other effects in the case of reactions in solution. We analyze dynamical effects both in the neighborhood of the transition state and far from it, together with the situation when quantum nuclear motion is central to the reaction, and we illustrate our discussion with various examples of enzymatic reactions. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

PubMed Central

Liu, Tian; Zhou, Yong; Chen, Lei; Chen, Wei; Liu, Lin; Shen, Xu; Zhang, Wenqing; Zhang, Jianzhen; Yang, Qing

2012-01-01

The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp490, Glu328, Val327 in OfHex1, and Trp358, Tyr131 and Ile179 in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu328 together with Trp490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K m for (GlcNAc)2 and a 42-fold increment in K i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu368 and Asp367) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu328 and Val327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes. PMID:23300622

A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry.

PubMed

Guy, Jodie E; Abreu, Isabel A; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-11-14

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.
A single mutation in the castor Δ9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

PubMed Central

Guy, Jodie E.; Abreu, Isabel A.; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-01-01

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by ≈2 × 103-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme. PMID:17088542
Isolation, purification, and characterization of AgUCGalT1, a galactosyltransferase involved in anthocyanin galactosylation in purple celery (Apium graveolens L.).

PubMed

Feng, Kai; Xu, Zhi-Sheng; Liu, Jie-Xia; Li, Jing-Wen; Wang, Feng; Xiong, Ai-Sheng

2018-06-01

This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins. In the present study, LC-MS data indicated that abundant cyanidin-based anthocyanins accumulated in the petioles of purple celery ('Nanxuan liuhe purple celery'). A gene encoding UDP-galactose: cyanidin 3-O-galactosyltransferase, namely AgUCGalT1, was isolated from purple celery and expressed in Escherichia coli BL21 (DE3). Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase (PSPG) motif. The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment. The recombinant AgUCGalT1 had an optimal enzyme activity at 35 °C and pH 8.0, and showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances, namely, peonidin, quercetin, and kaempferol. The expression levels of AgUCGalT1 were positively correlated with the total anthocyanin contents in purple and non-purple celery varieties. Crude enzymes extracted from purple celery exhibited glycosylation ability, whereas crude enzymes obtained from non-purple celery did not have this ability. This work provided evidence as a basis for investigations on the function of AgUCGalT1 in anthocyanin glycosylation in purple celery.
Characterization of a nitrilase and a nitrile hydratase from Pseudomonas sp. strain UW4 that converts indole-3-acetonitrile to indole-3-acetic acid.

PubMed

Duca, Daiana; Rose, David R; Glick, Bernard R

2014-08-01

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.
SYNCHRONIZATION OF HETEROGENEOUS OSCILLATORS UNDER NETWORK MODIFICATIONS: PERTURBATION AND OPTIMIZATION OF THE SYNCHRONY ALIGNMENT FUNCTION

PubMed Central

Taylor, Dane; Skardal, Per Sebastian; Sun, Jie

2016-01-01

Synchronization is central to many complex systems in engineering physics (e.g., the power-grid, Josephson junction circuits, and electro-chemical oscillators) and biology (e.g., neuronal, circadian, and cardiac rhythms). Despite these widespread applications—for which proper functionality depends sensitively on the extent of synchronization—there remains a lack of understanding for how systems can best evolve and adapt to enhance or inhibit synchronization. We study how network modifications affect the synchronization properties of network-coupled dynamical systems that have heterogeneous node dynamics (e.g., phase oscillators with non-identical frequencies), which is often the case for real-world systems. Our approach relies on a synchrony alignment function (SAF) that quantifies the interplay between heterogeneity of the network and of the oscillators and provides an objective measure for a system’s ability to synchronize. We conduct a spectral perturbation analysis of the SAF for structural network modifications including the addition and removal of edges, which subsequently ranks the edges according to their importance to synchronization. Based on this analysis, we develop gradient-descent algorithms to efficiently solve optimization problems that aim to maximize phase synchronization via network modifications. We support these and other results with numerical experiments. PMID:27872501
Immersion and dry scanner extensions for sub-10nm production nodes

NASA Astrophysics Data System (ADS)

Weichselbaum, Stefan; Bornebroek, Frank; de Kort, Toine; Droste, Richard; de Graaf, Roelof F.; van Ballegoij, Rob; Botter, Herman; McLaren, Matthew G.; de Boeij, Wim P.

2015-03-01

Progressing towards the 10nm and 7nm imaging node, pattern-placement and layer-to-layer overlay requirements keep on scaling down and drives system improvements in immersion (ArFi) and dry (ArF/KrF) scanners. A series of module enhancements in the NXT platform have been introduced; among others, the scanner is equipped with exposure stages with better dynamics and thermal control. Grid accuracy improvements with respect to calibration, setup, stability, and layout dependency tighten MMO performance and enable mix and match scanner operation. The same platform improvements also benefit focus control. Improvements in detectability and reproducibility of low contrast alignment marks enhance the alignment solution window for 10nm logic processes and beyond. The system's architecture allows dynamic use of high-order scanner optimization based on advanced actuators of projection lens and scanning stages. This enables a holistic optimization approach for the scanner, the mask, and the patterning process. Productivity scanner design modifications esp. stage speeds and optimization in metrology schemes provide lower layer costs for customers using immersion lithography as well as conventional dry technology. Imaging, overlay, focus, and productivity data is presented, that demonstrates 10nm and 7nm node litho-capability for both (immersion & dry) platforms.
On Multi-Dimensional Unstructured Mesh Adaption

NASA Technical Reports Server (NTRS)

Wood, William A.; Kleb, William L.

1999-01-01

Anisotropic unstructured mesh adaption is developed for a truly multi-dimensional upwind fluctuation splitting scheme, as applied to scalar advection-diffusion. The adaption is performed locally using edge swapping, point insertion/deletion, and nodal displacements. Comparisons are made versus the current state of the art for aggressive anisotropic unstructured adaption, which is based on a posteriori error estimates. Demonstration of both schemes to model problems, with features representative of compressible gas dynamics, show the present method to be superior to the a posteriori adaption for linear advection. The performance of the two methods is more similar when applied to nonlinear advection, with a difference in the treatment of shocks. The a posteriori adaption can excessively cluster points to a shock, while the present multi-dimensional scheme tends to merely align with a shock, using fewer nodes. As a consequence of this alignment tendency, an implementation of eigenvalue limiting for the suppression of expansion shocks is developed for the multi-dimensional distribution scheme. The differences in the treatment of shocks by the adaption schemes, along with the inherently low levels of artificial dissipation in the fluctuation splitting solver, suggest the present method is a strong candidate for applications to compressible gas dynamics.
A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.

PubMed

Shinozuka, Hiroshi; Cogan, Noel O I; Shinozuka, Maiko; Marshall, Alexis; Kay, Pippa; Lin, Yi-Han; Spangenberg, German C; Forster, John W

2015-04-11

Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.
The Impact of Structural Heterogeneity on Excitation-Inhibition Balance in Cortical Networks.

PubMed

Landau, Itamar D; Egger, Robert; Dercksen, Vincent J; Oberlaender, Marcel; Sompolinsky, Haim

2016-12-07

Models of cortical dynamics often assume a homogeneous connectivity structure. However, we show that heterogeneous input connectivity can prevent the dynamic balance between excitation and inhibition, a hallmark of cortical dynamics, and yield unrealistically sparse and temporally regular firing. Anatomically based estimates of the connectivity of layer 4 (L4) rat barrel cortex and numerical simulations of this circuit indicate that the local network possesses substantial heterogeneity in input connectivity, sufficient to disrupt excitation-inhibition balance. We show that homeostatic plasticity in inhibitory synapses can align the functional connectivity to compensate for structural heterogeneity. Alternatively, spike-frequency adaptation can give rise to a novel state in which local firing rates adjust dynamically so that adaptation currents and synaptic inputs are balanced. This theory is supported by simulations of L4 barrel cortex during spontaneous and stimulus-evoked conditions. Our study shows how synaptic and cellular mechanisms yield fluctuation-driven dynamics despite structural heterogeneity in cortical circuits. Copyright Â© 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
Magnetic Alignment of Block Copolymer Microdomains by Intrinsic Chain Anisotropy.

PubMed

Rokhlenko, Yekaterina; Gopinadhan, Manesh; Osuji, Chinedum O; Zhang, Kai; O'Hern, Corey S; Larson, Steven R; Gopalan, Padma; Majewski, Paweł W; Yager, Kevin G

2015-12-18

We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δχ, that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δχ≈2×10^{-8}. From field-dependent scattering data, we estimate that grains of ≈1.2 μm are present during alignment. These results demonstrate that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.
Multicolor microcontact printing of proteins on nanoporous surface for patterned immunoassay

NASA Astrophysics Data System (ADS)

Ng, Elaine; Gopal, Ashwini; Hoshino, Kazunori; Zhang, Xiaojing

2011-07-01

The large scale patterning of therapeutic proteins is a key to the efficient design, characterization, and production of biologics for cost effective, high throughput, and point-of-care detection and analysis system. We demonstrate an efficient method for protein deposition and adsorption on nanoporous silica substrates in specific patterns using a method called "micro-contact printing". Multiple color-tagged proteins can be printed through sequential application of such micro-patterning technique. Two groups of experiments were performed. In the first group, the protein stamp was aligned precisely with the printing sites, where the stamp was applied multiple times. Optimal conditions were identified for protein transfer and adsorption using the pore size of 4 nm and thickness of 30 nm porous silica thin film. In the second group, we demonstrate the patterning of two-color rabbit immunoglobin labeled with fluorescein isothiocyanate and tetramethyl rhodamine iso-thiocyanate on porous silica substrates that have a pore size 4 nm, porosity 57% and thickness of the porous layer 30 nm. A pair of protein stamps, with corresponding alignment markings and coupled patterns, were aligned and used to produce a two-colored stamp pattern of proteins on porous silica. Different colored proteins can be applied to exemplify the diverse protein composition within a sample. This method of multicolor microcontact printing can be used to perform a fluorescence-based patterned enzyme-linked immunosorbent assay to detect the presence of various proteins within a sample.
Making Enzyme Kinetics Dynamic via Simulation Software

ERIC Educational Resources Information Center

Potratz, Jeffrey P.

2017-01-01

An interactive classroom demonstration that enhances students' knowledge of steady-state and Michaelis-Menten enzyme kinetics is described. The instructor uses a free version of professional-quality KinTek Explorer simulation software and student input to construct dynamic versions of three static hallmark images commonly used to introduce enzyme…
Relationship between femtosecond-picosecond dynamics to enzyme catalyzed H-transfer

PubMed Central

Cheatum, Christopher M.; Kohen, Amnon

2015-01-01

At physiological temperatures, enzymes exhibit a broad spectrum of conformations, which interchange via thermally activated dynamics. These conformations are sampled differently in different complexes of the protein and its ligands, and the dynamics of exchange between these conformers depends on the mass of the group that is moving and the length scale of the motion, as well as restrictions imposed by the globular fold of the enzymatic complex. Many of these motions have been examined and their role in the enzyme function illuminated, yet most experimental tools applied so far have identified dynamics at time scales of seconds to nanoseconds, which are much slower than the time scale for H-transfer between two heavy atoms. This chemical conversion and other processes involving cleavage of covalent bonds occur on picosecond to femtosecond time scales, where slower processes mask both the kinetics and dynamics. Here we present a combination of kinetic and spectroscopic methods that may enable closer examination of the relationship between enzymatic C-H→C transfer and the dynamics of the active site environment at the chemically relevant time scale. These methods include kinetic isotope effects and their temperature dependence, which are used to study the kinetic nature of the H-transfer, and 2D IR spectroscopy, which is used to study the dynamics of transition-state- and ground-state-analog complexes. The combination of these tools is likely to provide a new approach to examine the protein dynamics that directly influence the chemical conversion catalyzed by enzymes. PMID:23539379
Taking directions: the role of microtubule-bound nucleation in the self-organization of the plant cortical array

NASA Astrophysics Data System (ADS)

Deinum, Eva E.; Tindemans, Simon H.; Mulder, Bela M.

2011-10-01

The highly aligned cortical microtubule array of interphase plant cells is a key regulator of anisotropic cell expansion. Recent computational and analytical work has shown that the non-equilibrium self-organization of this structure can be understood on the basis of experimentally observed collisional interactions between dynamic microtubules attached to the plasma membrane. Most of these approaches assumed that new microtubules are homogeneously and isotropically nucleated on the cortical surface. Experimental evidence, however, shows that nucleation mostly occurs from other microtubules and under specific relative angles. Here, we investigate the impact of directed microtubule-bound nucleations on the alignment process using computer simulations. The results show that microtubule-bound nucleations can increase the degree of alignment achieved, decrease the timescale of the ordering process and widen the regime of dynamic parameters for which the system can self-organize. We establish that the major determinant of this effect is the degree of co-alignment of the nucleations with the parent microtubule. The specific role of sideways branching nucleations appears to allow stronger alignment while maintaining a measure of overall spatial homogeneity. Finally, we investigate the suggestion that observed persistent rotation of microtubule domains can be explained through a handedness bias in microtubule-bound nucleations, showing that this is possible only for an extreme bias and over a limited range of parameters.
FtsZ rings and helices: physical mechanisms for the dynamic alignment of biopolymers in rod-shaped bacteria.

PubMed

Fischer-Friedrich, Elisabeth; Friedrich, Benjamin M; Gov, Nir S

2012-02-01

In many bacterial species, the protein FtsZ forms a cytoskeletal ring that marks the future division site and scaffolds the division machinery. In rod-shaped bacteria, most frequently membrane-attached FtsZ rings or ring fragments are reported and occasionally helices. By contrast, axial FtsZ clusters have never been reported. In this paper, we investigate theoretically how dynamic FtsZ aggregates align in rod-shaped bacteria. We study systematically different physical mechanisms that affect the alignment of FtsZ polymers using a computational model that relies on autocatalytic aggregation of FtsZ filaments at the membrane. Our study identifies a general tool kit of physical and geometrical mechanisms by which rod-shaped cells align biopolymer aggregates. Our analysis compares the relative impact of each mechanism on the circumferential alignment of FtsZ as observed in rod-shaped bacteria. We determine spontaneous curvature of FtsZ polymers and axial confinement of FtsZ on the membrane as the strongest factors. Including Min oscillations in our model, we find that these stabilize axial and helical clusters on short time scales, but promote the formation of an FtsZ ring at the cell middle at longer times. This effect could provide an explanation to the long standing puzzle of transiently observed oscillating FtsZ helices in Escherichia coli cells prior to cell division.
Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics

NASA Astrophysics Data System (ADS)

Linder, Mats; Johansson, Adam Johannes; Olsson, Tjelvar S. G.; Liebeschuetz, John; Brinck, Tore

2012-09-01

A novel computational Diels-Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus, is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels-Alderases with good results. The final designs in this work are predicted to have rate enhancements of ≈103-106 and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, `ideal' model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme ( T opt = 70 °C).
The Sulfur Oxygenase Reductase from the Mesophilic Bacterium Halothiobacillus neapolitanus Is a Highly Active Thermozyme

PubMed Central

Veith, Andreas; Botelho, Hugo M.; Kindinger, Florian; Gomes, Cláudio M.

2012-01-01

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere. PMID:22139503
Vertically aligned carbon nanofiber as nano-neuron interface for monitoring neural function.

PubMed

Yu, Zhe; McKnight, Timothy E; Ericson, M Nance; Melechko, Anatoli V; Simpson, Michael L; Morrison, Barclay

2012-05-01

Neural chips, which are capable of simultaneous multisite neural recording and stimulation, have been used to detect and modulate neural activity for almost thirty years. As neural interfaces, neural chips provide dynamic functional information for neural decoding and neural control. By improving sensitivity and spatial resolution, nano-scale electrodes may revolutionize neural detection and modulation at cellular and molecular levels as nano-neuron interfaces. We developed a carbon-nanofiber neural chip with lithographically defined arrays of vertically aligned carbon nanofiber electrodes and demonstrated its capability of both stimulating and monitoring electrophysiological signals from brain tissues in vitro and monitoring dynamic information of neuroplasticity. This novel nano-neuron interface may potentially serve as a precise, informative, biocompatible, and dual-mode neural interface for monitoring of both neuroelectrical and neurochemical activity at the single-cell level and even inside the cell. The authors demonstrate the utility of a neural chip with lithographically defined arrays of vertically aligned carbon nanofiber electrodes. The new device can be used to stimulate and/or monitor signals from brain tissue in vitro and for monitoring dynamic information of neuroplasticity both intracellularly and at the single cell level including neuroelectrical and neurochemical activities. Copyright © 2012 Elsevier Inc. All rights reserved.
Topography on a subcellular scale modulates cellular adhesions and actin stress fiber dynamics in tumor associated fibroblasts

NASA Astrophysics Data System (ADS)

Azatov, Mikheil; Sun, Xiaoyu; Suberi, Alexandra; Fourkas, John T.; Upadhyaya, Arpita

2017-12-01

Cells can sense and adapt to mechanical properties of their environment. The local geometry of the extracellular matrix, such as its topography, has been shown to modulate cell morphology, migration, and proliferation. Here we investigate the effect of micro/nanotopography on the morphology and cytoskeletal dynamics of human pancreatic tumor-associated fibroblast cells (TAFs). We use arrays of parallel nanoridges with variable spacings on a subcellular scale to investigate the response of TAFs to the topography of their environment. We find that cell shape and stress fiber organization both align along the direction of the nanoridges. Our analysis reveals a strong bimodal relationship between the degree of alignment and the spacing of the nanoridges. Furthermore, focal adhesions align along ridges and form preferentially on top of the ridges. Tracking actin stress fiber movement reveals enhanced dynamics of stress fibers on topographically patterned surfaces. We find that components of the actin cytoskeleton move preferentially along the ridges with a significantly higher velocity along the ridges than on a flat surface. Our results suggest that a complex interplay between the actin cytoskeleton and focal adhesions coordinates the cellular response to micro/nanotopography.
Genome alignment with graph data structures: a comparison

PubMed Central

2014-01-01

Background Recent advances in rapid, low-cost sequencing have opened up the opportunity to study complete genome sequences. The computational approach of multiple genome alignment allows investigation of evolutionarily related genomes in an integrated fashion, providing a basis for downstream analyses such as rearrangement studies and phylogenetic inference. Graphs have proven to be a powerful tool for coping with the complexity of genome-scale sequence alignments. The potential of graphs to intuitively represent all aspects of genome alignments led to the development of graph-based approaches for genome alignment. These approaches construct a graph from a set of local alignments, and derive a genome alignment through identification and removal of graph substructures that indicate errors in the alignment. Results We compare the structures of commonly used graphs in terms of their abilities to represent alignment information. We describe how the graphs can be transformed into each other, and identify and classify graph substructures common to one or more graphs. Based on previous approaches, we compile a list of modifications that remove these substructures. Conclusion We show that crucial pieces of alignment information, associated with inversions and duplications, are not visible in the structure of all graphs. If we neglect vertex or edge labels, the graphs differ in their information content. Still, many ideas are shared among all graph-based approaches. Based on these findings, we outline a conceptual framework for graph-based genome alignment that can assist in the development of future genome alignment tools. PMID:24712884

Alpha LAMP Integration Facility

NASA Technical Reports Server (NTRS)

Oshiro, Richard; Sowers, Dennis; Gargiulo, Joe; Mcgahey, Mark

1994-01-01

This paper describes the activity recently completed to meet the simulated space environment requirements for the ground-based testing of an integrated Space Based Laser (SBL) system experiment. The need to maintain optical alignment in the challenging dynamic environment of the pressure recovery system required to simulate space dominated the design requirements. A robust system design was established which minimized the total program costs, most notably by reducing the cost of integrating the components of the experiment. The components of the experiment are integrated on an optical bench in a clean area adjacent to the vacuum chamber and moved on air bearings into the chamber for testing.
Space Technology 5 Multipoint Observations of Temporal and Spatial Variability of Field-Aligned Currents

NASA Technical Reports Server (NTRS)

Le, G.; Wang, Y.; Slavin, J. A.; Strangeway, R. L.

2009-01-01

Space Technology 5 (ST5) is a constellation mission consisting of three microsatellites. It provides the first multipoint magnetic field measurements in low Earth orbit, which enables us to separate spatial and temporal variations. In this paper, we present a study of the temporal variability of field-aligned currents using the ST5 data. We examine the field-aligned current observations during and after a geomagnetic storm and compare the magnetic field profiles at the three spacecraft. The multipoint data demonstrate that mesoscale current structures, commonly embedded within large-scale current sheets, are very dynamic with highly variable current density and/or polarity in approx.10 min time scales. On the other hand, the data also show that the time scales for the currents to be relatively stable are approx.1 min for mesoscale currents and approx.10 min for large-scale currents. These temporal features are very likely associated with dynamic variations of their charge carriers (mainly electrons) as they respond to the variations of the parallel electric field in auroral acceleration region. The characteristic time scales for the temporal variability of mesoscale field-aligned currents are found to be consistent with those of auroral parallel electric field.
Disentangling the Role of Entanglement Density and Molecular Alignment in the Mechanical Response of Glassy Polymers

NASA Astrophysics Data System (ADS)

O'Connor, Thomas; Robbins, Mark

Glassy polymers are a ubiquitous part of modern life, but much about their mechanical properties remains poorly understood. Since chains in glassy states are hindered from exploring their conformational entropy, they can't be understood with common entropic network models. Additionally, glassy states are highly sensitive to material history and nonequilibrium distributions of chain alignment and entanglement can be produced during material processing. Understanding how these far-from equilibrium states impact mechanical properties is analytically challenging but essential to optimizing processing methods. We use molecular dynamics simulations to study the yield and strain hardening of glassy polymers as separate functions of the degree of molecular alignment and inter-chain entanglement. We vary chain alignment and entanglement with three different preparation protocols that mimic common processing conditions in and out of solution. We compare our results to common mechanical models of amorphous polymers and assess their applicability to different experimental processing conditions. This research was performed within the Center for Materials in Extreme Dynamic Environments (CMEDE) under the Hopkins Extreme Materials Institute at Johns Hopkins University. Financial support was provided by Grant W911NF-12-2-0022.
Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences.

PubMed

Arend, J; Warzecha, H; Stöckigt, J

2000-01-01

Plant cell suspension cultures of Rauvolfia are able to produce a high amount of arbutin by glucosylation of exogenously added hydroquinone. A four step purification procedure using anion exchange, hydrophobic interaction, hydroxyapatite-chromatography and chromatofocusing delivered in a yield of 0.5%, an approximately 390 fold enrichment of the involved glucosyltransferase. SDS-PAGE showed a M(r) for the enzyme of 52 kDa. Proteolysis of the pure enzyme with endoproteinase LysC revealed six peptide fragments with 9-23 amino acids which were sequenced. Sequence alignment of the six peptides showed high homologies to glycosyltransferases from other higher plants.
Assembly of synthetic cellulose I.

PubMed

Lee, J H; Brown, R M; Kuga, S; Shoda, S; Kobayashi, S

1994-08-02

Cellulose microfibrils with an electron diffraction pattern characteristic of crystalline native cellulose I have been assembled abiotically by means of a cellulase-catalyzed polymerization of beta-cellobiosyl fluoride substrate monomer in acetonitrile/acetate buffer. Substantial purification of the Trichoderma viride cellulase enzyme was found to be essential for the formation of the synthetic cellulose I allomorph. Assembly of synthetic cellulose I appears to be a result of a micellar aggregation of the partially purified enzyme and the substrate in an organic/aqueous solvent system favoring the alignment of glucan chains with the same polarity and extended chain conformation, resulting in crystallization to form the metastable cellulose I allomorph.
Tree decomposition based fast search of RNA structures including pseudoknots in genomes.

PubMed

Song, Yinglei; Liu, Chunmei; Malmberg, Russell; Pan, Fangfang; Cai, Liming

2005-01-01

Searching genomes for RNA secondary structure with computational methods has become an important approach to the annotation of non-coding RNAs. However, due to the lack of efficient algorithms for accurate RNA structure-sequence alignment, computer programs capable of fast and effectively searching genomes for RNA secondary structures have not been available. In this paper, a novel RNA structure profiling model is introduced based on the notion of a conformational graph to specify the consensus structure of an RNA family. Tree decomposition yields a small tree width t for such conformation graphs (e.g., t = 2 for stem loops and only a slight increase for pseudo-knots). Within this modelling framework, the optimal alignment of a sequence to the structure model corresponds to finding a maximum valued isomorphic subgraph and consequently can be accomplished through dynamic programming on the tree decomposition of the conformational graph in time O(k(t)N(2)), where k is a small parameter; and N is the size of the projiled RNA structure. Experiments show that the application of the alignment algorithm to search in genomes yields the same search accuracy as methods based on a Covariance model with a significant reduction in computation time. In particular; very accurate searches of tmRNAs in bacteria genomes and of telomerase RNAs in yeast genomes can be accomplished in days, as opposed to months required by other methods. The tree decomposition based searching tool is free upon request and can be downloaded at our site h t t p ://w.uga.edu/RNA-informatics/software/index.php.
Molecular determinants of enzyme cold adaptation: comparative structural and computational studies of cold- and warm-adapted enzymes.

PubMed

Papaleo, Elena; Tiberti, Matteo; Invernizzi, Gaetano; Pasi, Marco; Ranzani, Valeria

2011-11-01

The identification of molecular mechanisms underlying enzyme cold adaptation is a hot-topic both for fundamental research and industrial applications. In the present contribution, we review the last decades of structural computational investigations on cold-adapted enzymes in comparison to their warm-adapted counterparts. Comparative sequence and structural studies allow the definition of a multitude of adaptation strategies. Different enzymes carried out diverse mechanisms to adapt to low temperatures, so that a general theory for enzyme cold adaptation cannot be formulated. However, some common features can be traced in dynamic and flexibility properties of these enzymes, as well as in their intra- and inter-molecular interaction networks. Interestingly, the current data suggest that a family-centered point of view is necessary in the comparative analyses of cold- and warm-adapted enzymes. In fact, enzymes belonging to the same family or superfamily, thus sharing at least the three-dimensional fold and common features of the functional sites, have evolved similar structural and dynamic patterns to overcome the detrimental effects of low temperatures.
Electrostatically Induced Carbon Nanotube Alignment for Polymer Composite Applications

NASA Astrophysics Data System (ADS)

Chapkin, Wesley Aaron

We have developed a non-invasive technique utilizing polarized Raman spectroscopy to measure changes in carbon nanotube (CNT) alignment in situ and in real time in a polymer matrix. With this technique, we have confirmed the prediction of faster alignment for CNTs in higher electric fields. Real-time polarized Raman spectroscopy also allows us to demonstrate the loss of CNT alignment that occurs after the electric field is removed, which reveals the need for fast polymerization steps or the continued application of the aligning force during polymerization to lock in CNT alignment. Through a study on the effect of polymer viscosity on the rate of CNT alignment, we have determined that shear viscosity serves as the controlling mechanism for CNT rotation. This finding matches literature modeling of rigid rod mobility in a polymer melt and demonstrates that the rotational mobility of CNTs can be explained by a continuum model even though the diameters of single-walled CNTs are 1-2 nm. The viscosity dependence indicates that the manipulation of temperature (and indirectly viscosity) will have a direct effect on the rate of CNT alignment, which could prove useful in expediting the manufacturing of CNT-reinforced composites cured at elevated temperatures. Using real-time polarized Raman spectroscopy, we also demonstrate that electric fields of various strengths lead not only to different speeds of CNT rotation but also to different degrees of alignment. We hypothesize that this difference in achievable alignment results from discrete populations of nanotubes based on their length. The results are then explained by balancing the alignment energy for a given electric field strength with the randomizing thermal energy of the system. By studying the alignment dynamics of different CNT length distributions, we show that different degrees of alignment achieved as a function of the applied electric field strength are directly related to the square of the nanotube length. This finding matches an electrostatic potential energy model for CNT rotation. Lastly, we investigate the effects of conductive carbon fibers on electrostatically induced alignment of CNTs within carbon fiber composites. The relative electric field strength throughout the composite is modeled using COMSOL Multiphysics. We show the ability to generate enhanced electric field gradients within the gaps between carbon fibers for various fiber orientations. Using polarized Raman spectroscopy, increased levels of CNT alignment are observed between carbon fiber tows, which is consistent with the modeled higher electric field strengths in these regions. These findings could potentially lead to the development of carbon fiber composites with CNT additions that selectively enhance the composite properties outside the carbon fiber interphase in the neat epoxy.
A Novel Adaptive H∞ Filtering Method with Delay Compensation for the Transfer Alignment of Strapdown Inertial Navigation Systems.

PubMed

Lyu, Weiwei; Cheng, Xianghong

2017-11-28

Transfer alignment is always a key technology in a strapdown inertial navigation system (SINS) because of its rapidity and accuracy. In this paper a transfer alignment model is established, which contains the SINS error model and the measurement model. The time delay in the process of transfer alignment is analyzed, and an H∞ filtering method with delay compensation is presented. Then the H∞ filtering theory and the robust mechanism of H∞ filter are deduced and analyzed in detail. In order to improve the transfer alignment accuracy in SINS with time delay, an adaptive H∞ filtering method with delay compensation is proposed. Since the robustness factor plays an important role in the filtering process and has effect on the filtering accuracy, the adaptive H∞ filter with delay compensation can adjust the value of robustness factor adaptively according to the dynamic external environment. The vehicle transfer alignment experiment indicates that by using the adaptive H∞ filtering method with delay compensation, the transfer alignment accuracy and the pure inertial navigation accuracy can be dramatically improved, which demonstrates the superiority of the proposed filtering method.
Direct-write 3D printing of composite materials with magnetically aligned discontinuous reinforcement

NASA Astrophysics Data System (ADS)

Martin, Joshua J.; Caunter, Andrew; Dendulk, Amy; Goodrich, Scott; Pembroke, Ryan; Shores, Dan; Erb, Randall M.

2017-05-01

Three-dimensional (3D) printing of fiber reinforced composites represents an enabling technology that may bring toughness and specific strength to complex parts. Recently, direct-write 3D printing has been offered as a promising route to manufacturing fiber reinforced composites that show high specific strength. These approaches primarily rely on the use of shear-alignment during the extrusion process to align fibers along the printing direction. Shear alignment prevents fibers from being oriented along principle stress directions of the final designed part. This paper describes a new direct-write style 3D printing system that incorporates magnetic fields to actively control the orientation of reinforcing fibers during the printing of fiber reinforced composites. Such a manufacturing system is fraught with complications from the high shear dominated alignment experienced by the fibers during extrusion to the slow magnetic alignment dynamics of fibers in viscous media. Here we characterize these issues and suggest effective operating windows in which magnetic alignment is a viable approach to orienting reinforcing particles during direct-write 3D printing.
Exact calculation of distributions on integers, with application to sequence alignment.

PubMed

Newberg, Lee A; Lawrence, Charles E

2009-01-01

Computational biology is replete with high-dimensional discrete prediction and inference problems. Dynamic programming recursions can be applied to several of the most important of these, including sequence alignment, RNA secondary-structure prediction, phylogenetic inference, and motif finding. In these problems, attention is frequently focused on some scalar quantity of interest, a score, such as an alignment score or the free energy of an RNA secondary structure. In many cases, score is naturally defined on integers, such as a count of the number of pairing differences between two sequence alignments, or else an integer score has been adopted for computational reasons, such as in the test of significance of motif scores. The probability distribution of the score under an appropriate probabilistic model is of interest, such as in tests of significance of motif scores, or in calculation of Bayesian confidence limits around an alignment. Here we present three algorithms for calculating the exact distribution of a score of this type; then, in the context of pairwise local sequence alignments, we apply the approach so as to find the alignment score distribution and Bayesian confidence limits.
F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations

PubMed Central

Mancini, Giordano; Zazza, Costantino

2015-01-01

The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization. PMID:26415031
F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations.

PubMed

Mancini, Giordano; Zazza, Costantino

2015-01-01

The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.
Pair aligning improved motility of Quincke rollers.

PubMed

Lu, Shi Qing; Zhang, Bing Yue; Zhang, Zhi Chao; Shi, Yan; Zhang, Tian Hui

2018-06-06

Density-dependent speed is studied in a two-dimensional active colloid in which the colloidal particles are propelled by an external electric field via a Quincke rotation. Above the critcal electric field, dense dynamic clusters form spotaneously, in which the particles are highly aligned in velocity and move much faster than isolated units. Detailed observations on pair collision reveal that the alignment of velocity is induced by the long-ranged hydrodynamic interactions and the improvement of speed in the clusters arises from pair aligning in which two particles are closely paired and rotate synchronically. In the aligning state, the short-range in-plane dipole-dipole attraction enhances the rotation torque and gives rises to a larger rolling speed. The pair aligning becomes difficult and unstable at high electric field where the normal dipole-dipole repulsion becomes dominant. As a consequence, the dependence of speed on density becomes weak increasingly upon the increase of the electric field. This result offers an interpretation for the discrepancy between our and previous observations on Quincke rollers.
Active and Passive Radiative Transfer Modeling with Preferentially-Aligned Particles

NASA Technical Reports Server (NTRS)

Adams, Ian Stuart

2017-01-01

The fluid dynamics of falling hydrometeors often results in preferential orientations that can affect both the intensity and polarization of electromagnetic radiation. In order to properly interpret remote sensing observations of ice and snow, such alignments should be considered when constructing databases of scattering particles; however, the inclusion of aligned particles increases the complexity of the scattering data. To demonstrate the use of scattering properties of preferentially-aligned particles, millimeter-wave brightness temperatures and radar observables, including reflectivity and linear depolarization ratio, are modeled using the Atmospheric Radiative Transfer Simulator (ARTS). The necessary scattering parameters for vector radiative transfer, particularly with respect to ARTS, are reviewed, and the exploitation of particle symmetries, as well as scattering reciprocity relationships, are detailed.
Spacecraft alignment estimation. [for onboard sensors

NASA Technical Reports Server (NTRS)

Shuster, Malcolm D.; Bierman, Gerald J.

1988-01-01

A numerically well-behaved factorized methodology is developed for estimating spacecraft sensor alignments from prelaunch and inflight data without the need to compute the spacecraft attitude or angular velocity. Such a methodology permits the estimation of sensor alignments (or other biases) in a framework free of unknown dynamical variables. In actual mission implementation such an algorithm is usually better behaved than one that must compute sensor alignments simultaneously with the spacecraft attitude, for example by means of a Kalman filter. In particular, such a methodology is less sensitive to data dropouts of long duration, and the derived measurement used in the attitude-independent algorithm usually makes data checking and editing of outliers much simpler than would be the case in the filter.
Experimental assessment of the importance of amino acid positions identified by an entropy-based correlation analysis of multiple-sequence alignments.

PubMed

Dietrich, Susanne; Borst, Nadine; Schlee, Sandra; Schneider, Daniel; Janda, Jan-Oliver; Sterner, Reinhard; Merkl, Rainer

2012-07-17

The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e., a large number of significant correlations in which k is involved. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r mutant proteins were generated by replacing nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal [conn(k) = 11]. For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability decreases were much less severe. Our findings demonstrate that positions with high conn(k) values have an increased probability of being important for enzyme function or stability.
Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

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

Yu, Hao; Dranchak, Patricia; Li, Zhiru

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanismmore » placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.« less
Energy Level Alignment at Aqueous GaN and ZnO Interfaces

NASA Astrophysics Data System (ADS)

Hybertsen, Mark S.; Kharche, Neerav; Muckerman, James T.

2014-03-01

Electronic energy level alignment at semiconductor-electrolyte interfaces is fundamental to electrochemical activity. Motivated in particular by the search for new materials that can be more efficient for photocatalysis, we develop a first principles method to calculate this alignment at aqueous interfaces and demonstrate it for the specific case of non-polar GaN and ZnO interfaces with water. In the first step, density functional theory (DFT) based molecular dynamics is used to sample the physical interface structure and to evaluate the electrostatic potential step at the interface. In the second step, the GW approach is used to evaluate the reference electronic energy level separately in the bulk semiconductor (valence band edge energy) and in bulk water (the 1b1 energy level), relative to the internal electrostatic energy reference. Use of the GW approach naturally corrects for errors inherent in the use of Kohn-Sham energy eigenvalues to approximate the electronic excitation energies in each material. With this predicted interface alignment, specific redox levels in water, with potentials known relative to the 1b1 level, can then be compared to the semiconductor band edge positions. Our results will be discussed in the context of experiments in which photoexcited GaN and ZnO drive the hydrogen evolution reaction. Research carried out at Brookhaven National Laboratory under Contract No. DE-AC02-98CH10886 with the U.S. Department of Energy.
From dynamic measurements of photosynthesis in a living plant to sunlight transformation into electricity.

PubMed

Flexer, Victoria; Mano, Nicolas

2010-02-15

We propose here a new method for the direct and continuous measurement of O(2) and glucose generated during photosynthesis. Our system is based on amperometric enzyme biosensors comprising immobilized redox enzymes (glucose oxidase (GOx) and bilirubin oxidase (BOD)) and redox hydrogels "wiring" the enzyme reaction centers to electrodes. We found that these electrodes, implanted into a living plant, responded in real time to visible light as an external stimulus triggering photosynthesis. They proved to be highly selective and fast enough and may be a valuable tool in understanding photosynthesis kinetics. Furthermore, we demonstrate that with our electrodes we could harvest glucose and O(2) produced during photosynthesis to produce energy, transforming sunlight into electricity in a simple, green, renewable, and sustainable way.

Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

PubMed

Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

2014-01-01

Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.
Comparative Analysis of Mass Spectral Similarity Measures on Peak Alignment for Comprehensive Two-Dimensional Gas Chromatography Mass Spectrometry

PubMed Central

2013-01-01

Peak alignment is a critical procedure in mass spectrometry-based biomarker discovery in metabolomics. One of peak alignment approaches to comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) data is peak matching-based alignment. A key to the peak matching-based alignment is the calculation of mass spectral similarity scores. Various mass spectral similarity measures have been developed mainly for compound identification, but the effect of these spectral similarity measures on the performance of peak matching-based alignment still remains unknown. Therefore, we selected five mass spectral similarity measures, cosine correlation, Pearson's correlation, Spearman's correlation, partial correlation, and part correlation, and examined their effects on peak alignment using two sets of experimental GC×GC-MS data. The results show that the spectral similarity measure does not affect the alignment accuracy significantly in analysis of data from less complex samples, while the partial correlation performs much better than other spectral similarity measures when analyzing experimental data acquired from complex biological samples. PMID:24151524
Modeling the Response of Soil Organic Matter Decomposition to Warming: Effects of Dynamical Enzyme Productivity and Nuanced Representation of Respiration.

NASA Astrophysics Data System (ADS)

Sihi, D.; Gerber, S.; Inglett, K. S.; Inglett, P.

2014-12-01

Recent development in modeling soil organic carbon (SOC) decomposition includes the explicit incorporation of enzyme and microbial dynamics. A characteristic of these models is a feedback between substrate and consumers which is absent in traditional first order decay models. Second, microbial decomposition models incorporate carbon use efficiency (CUE) as a function of temperature which proved to be critical to prediction of SOC with warming. Our main goal is to explore microbial decomposition models with respect to responses of microbes to enzyme activity, costs to enzyme production, and to incorporation of growth vs. maintenance respiration. In order to simplify the modeling setup we assumed quick adjustment of enzyme activity and depolymerized carbon to microbial and SOC pools. Enzyme activity plays an important role to decomposition if its production is scaled to microbial biomass. In fact if microbes are allowed to optimize enzyme productivity the microbial enzyme model becomes unstable. Thus if the assumption of enzyme productivity is relaxed, other limiting factors must come into play. To stabilize the model, we account for two feedbacks that include cost of enzyme production and diminishing return of depolymerization with increasing enzyme concentration and activity. These feedback mechanisms caused the model to behave in a similar way to traditional, first order decay models. Most importantly, we found, that under warming, the changes in SOC carbon were more severe in enzyme synthesis is costly. In turn, carbon use efficiency (CUE) and its dynamical response to temperature is mainly determined by 1) the rate of turnover of microbes 2) the partitioning of dead microbial matter into different quality pools, and 3) and whether growth, maintenance respiration and microbial death rate have distinct responses to changes in temperature. Abbreviations: p: decay of enzyme, g: coefficient for growth respiration, : fraction of material from microbial turnover that enters the DOC pool, loss of C scaled to microbial mass, half saturation constant.
Relative impact of uniaxial alignment vs. form-induced stress on differentiation of human adipose derived stem cells

PubMed Central

Huang, Samuel; Li, Julie Yi-Shuan; Chien, Shu; Zhang, Kang; Chen, Shaochen

2013-01-01

ADSCs are a great cell source for tissue engineering and regenerative medicine. However, the development of methods to appropriately manipulate these cells in vitro remains a challenge. Here the proliferation and differentiation of ADSCs on microfabricated surfaces with varying geometries were investigated. To create the patterned substrates, a maskless biofabrication method was developed based on dynamic optical projection stereolithography. Proliferation and early differentiation of ADSCs were compared across three distinct multicellular patterns, namely stripes (ST), symmetric fork (SF), and asymmetric fork (AF). The ST pattern was designed for uniaxial cell alignment while the SF and AF pattern were designed with altered cell directionality to different extents. The SF and AF patterns generated similar levels of regional peak stress, which were both significantly higher than those within the ST pattern. No significant difference in ADSC proliferation was observed among the three patterns. In comparison to the ST pattern, higher peak stress levels of the SF and AF patterns were associated with up-regulation of the chondrogenic and osteogenic markers SOX9 and RUNX2. Interestingly, uniaxial cell alignment in the ST pattern seemed to increase the expression of SM22α and smooth muscle α-actin, suggesting an early smooth muscle lineage progression. These results indicate that geometric cues that promote uniaxial alignment might be more potent for myogenesis than those with increased peak stress. Overall, the use of these patterned geometric cues for modulating cell alignment and form-induced stress can serve as a powerful and versatile technique towards controlling differentiation in ADSCs. PMID:24060419
Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

PubMed Central

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao

2017-01-01

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (−)-Vince lactam. PMID:29054871
Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one).

PubMed

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao; Zheng, Guojun

2018-01-01

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (-)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (-)-Vince lactam. Copyright © 2017 American Society for Microbiology.
Alignment of time-resolved data from high throughput experiments.

PubMed

Abidi, Nada; Franke, Raimo; Findeisen, Peter; Klawonn, Frank

2016-12-01

To better understand the dynamics of the underlying processes in cells, it is necessary to take measurements over a time course. Modern high-throughput technologies are often used for this purpose to measure the behavior of cell products like metabolites, peptides, proteins, [Formula: see text]RNA or mRNA at different points in time. Compared to classical time series, the number of time points is usually very limited and the measurements are taken at irregular time intervals. The main reasons for this are the costs of the experiments and the fact that the dynamic behavior usually shows a strong reaction and fast changes shortly after a stimulus and then slowly converges to a certain stable state. Another reason might simply be missing values. It is common to repeat the experiments and to have replicates in order to carry out a more reliable analysis. The ideal assumptions that the initial stimulus really started exactly at the same time for all replicates and that the replicates are perfectly synchronized are seldom satisfied. Therefore, there is a need to first adjust or align the time-resolved data before further analysis is carried out. Dynamic time warping (DTW) is considered as one of the common alignment techniques for time series data with equidistant time points. In this paper, we modified the DTW algorithm so that it can align sequences with measurements at different, non-equidistant time points with large gaps in between. This type of data is usually known as time-resolved data characterized by irregular time intervals between measurements as well as non-identical time points for different replicates. This new algorithm can be easily used to align time-resolved data from high-throughput experiments and to come across existing problems such as time scarcity and existing noise in the measurements. We propose a modified method of DTW to adapt requirements imposed by time-resolved data by use of monotone cubic interpolation splines. Our presented approach provides a nonlinear alignment of two sequences that neither need to have equi-distant time points nor measurements at identical time points. The proposed method is evaluated with artificial as well as real data. The software is available as an R package tra (Time-Resolved data Alignment) which is freely available at: http://public.ostfalia.de/klawonn/tra.zip .
Structured crowding and its effects on enzyme catalysis.

PubMed

Ma, Buyong; Nussinov, Ruth

2013-01-01

Macromolecular crowding decreases the diffusion rate, shifts the equilibrium of protein-protein and protein-substrate interactions, and changes protein conformational dynamics. Collectively, these effects contribute to enzyme catalysis. Here we describe how crowding may bias the conformational change and dynamics of enzyme populations and in this way affect catalysis. Crowding effects have been studied using artificial crowding agents and in vivo-like environments. These studies revealed a correlation between protein dynamics and function in the crowded environment. We suggest that crowded environments be classified into uniform crowding and structured crowding. Uniform crowding represents random crowding conditions created by synthetic particles with a narrow size distribution. Structured crowding refers to the highly coordinated cellular environment, where proteins and other macromolecules are clustered and organized. In structured crowded environments the perturbation of protein thermal stability may be lower; however, it may still be able to modulate functions effectively and dynamically. Dynamic, allosteric enzymes could be more sensitive to cellular perturbations if their free energy landscape is flatter around the native state; on the other hand, if their free energy landscape is rougher, with high kinetic barriers separating deep minima, they could be more robust. Above all, cells are structured; and this holds both for the cytosol and for the membrane environment. The crowded environment is organized, which limits the search, and the crowders are not necessarily inert. More likely, they too transmit allosteric effects, and as such play important functional roles. Overall, structured cellular crowding may lead to higher enzyme efficiency and specificity.
Is multiple-sequence alignment required for accurate inference of phylogeny?

PubMed

Höhl, Michael; Ragan, Mark A

2007-04-01

The process of inferring phylogenetic trees from molecular sequences almost always starts with a multiple alignment of these sequences but can also be based on methods that do not involve multiple sequence alignment. Very little is known about the accuracy with which such alignment-free methods recover the correct phylogeny or about the potential for increasing their accuracy. We conducted a large-scale comparison of ten alignment-free methods, among them one new approach that does not calculate distances and a faster variant of our pattern-based approach; all distance-based alignment-free methods are freely available from http://www.bioinformatics.org.au (as Python package decaf+py). We show that most methods exhibit a higher overall reconstruction accuracy in the presence of high among-site rate variation. Under all conditions that we considered, variants of the pattern-based approach were significantly better than the other alignment-free methods. The new pattern-based variant achieved a speed-up of an order of magnitude in the distance calculation step, accompanied by a small loss of tree reconstruction accuracy. A method of Bayesian inference from k-mers did not improve on classical alignment-free (and distance-based) methods but may still offer other advantages due to its Bayesian nature. We found the optimal word length k of word-based methods to be stable across various data sets, and we provide parameter ranges for two different alphabets. The influence of these alphabets was analyzed to reveal a trade-off in reconstruction accuracy between long and short branches. We have mapped the phylogenetic accuracy for many alignment-free methods, among them several recently introduced ones, and increased our understanding of their behavior in response to biologically important parameters. In all experiments, the pattern-based approach emerged as superior, at the expense of higher resource consumption. Nonetheless, no alignment-free method that we examined recovers the correct phylogeny as accurately as does an approach based on maximum-likelihood distance estimates of multiply aligned sequences.
Gibbs Free Energy of Hydrolytic Water Molecule in Acyl-Enzyme Intermediates of a Serine Protease: A Potential Application for Computer-Aided Discovery of Mechanism-Based Reversible Covalent Inhibitors.

PubMed

Masuda, Yosuke; Yamaotsu, Noriyuki; Hirono, Shuichi

2017-01-01

In order to predict the potencies of mechanism-based reversible covalent inhibitors, the relationships between calculated Gibbs free energy of hydrolytic water molecule in acyl-trypsin intermediates and experimentally measured catalytic rate constants (k cat ) were investigated. After obtaining representative solution structures by molecular dynamics (MD) simulations, hydration thermodynamics analyses using WaterMap™ were conducted. Consequently, we found for the first time that when Gibbs free energy of the hydrolytic water molecule was lower, logarithms of k cat were also lower. The hydrolytic water molecule with favorable Gibbs free energy may hydrolyze acylated serine slowly. Gibbs free energy of hydrolytic water molecule might be a useful descriptor for computer-aided discovery of mechanism-based reversible covalent inhibitors of hydrolytic enzymes.
Surface tension-driven self-alignment.

PubMed

Mastrangeli, Massimo; Zhou, Quan; Sariola, Veikko; Lambert, Pierre

2017-01-04

Surface tension-driven self-alignment is a passive and highly-accurate positioning mechanism that can significantly simplify and enhance the construction of advanced microsystems. After years of research, demonstrations and developments, the surface engineering and manufacturing technology enabling capillary self-alignment has achieved a degree of maturity conducive to a successful transfer to industrial practice. In view of this transition, a broad and accessible review of the physics, material science and applications of capillary self-alignment is presented. Statics and dynamics of the self-aligning action of deformed liquid bridges are explained through simple models and experiments, and all fundamental aspects of surface patterning and conditioning, of choice, deposition and confinement of liquids, and of component feeding and interconnection to substrates are illustrated through relevant applications in micro- and nanotechnology. A final outline addresses remaining challenges and additional extensions envisioned to further spread the use and fully exploit the potential of the technique.
Magnetic alignment of block copolymer microdomains by intrinsic chain anisotropy

DOE PAGES

Rokhlenko, Yekaterina; Yager, Kevin G.; Gopinadhan, Manesh; ...

2015-12-18

We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δ χ, that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δ χ ≈ 2×10 –8. From field-dependent scattering data, we estimate that grains of ≈ 1.2 μm are present during alignment. Furthermore, these results demonstratemore » that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.« less
Magnetic alignment of block copolymer microdomains by intrinsic chain anisotropy

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

Rokhlenko, Yekaterina; Yager, Kevin G.; Gopinadhan, Manesh

We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δ χ, that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δ χ ≈ 2×10 –8. From field-dependent scattering data, we estimate that grains of ≈ 1.2 μm are present during alignment. Furthermore, these results demonstratemore » that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.« less
Simultaneous glucose production from cellulose and fouling reduction using a magnetic responsive membrane reactor with superparamagnetic nanoparticles carrying cellulolytic enzymes.

PubMed

Gebreyohannes, Abaynesh Yihdego; Dharmjeet, Madhav; Swusten, Tom; Mertens, Matthias; Verspreet, Joran; Verbiest, Thierry; Courtin, Christophe M; Vankelecom, Ivo F J

2018-05-02

This work aimed at investigating simultaneous hydrolysis of cellulose and in-situ foulant degradation in a cellulose fed superparamagnetic biocatalytic membrane reactor (BMR SP ). In this reactor, a dynamic layer of superparamagnetic bionanocomposites with immobilized cellulolytic enzymes were reversibly immobilized on superparamagnetic polymeric membrane using an external magnetic field. The formation of a dynamic layer of bionanocomposites on the membrane helped to prevent direct membrane-foulant interaction. Due to in-situ biocatalysis, there was limited filtration resistance. Simultaneous separation of the product helped to avoid enzyme product inhibition, achieve constant reaction rate over time and 50% higher enzyme efficiency than batch reactor. Stable enzyme immobilization and the ability to keep enzyme in the system for long period helped to achieve continuous productivity at very low enzyme but high solid loading, while also reducing the extent of membrane fouling. Hence, the BMR SP paves a path for sustainable production of bioethanol from the cheaply available lignocellulose. Copyright © 2018 Elsevier Ltd. All rights reserved.
A Framework for Evaluating and Enhancing Alignment in Self-Regulated Learning Research

PubMed Central

Dent, Amy L.; Hoyle, Rick H.

2015-01-01

We discuss the articles of this special issue with reference to an important yet previously only implicit dimension of study quality: alignment across the theoretical and methodological decisions that collectively define an approach to self-regulated learning. Integrating and extending work by leaders in the field, we propose a framework for evaluating alignment in the way self-regulated learning research is both conducted and reported. Within this framework, the special issue articles provide a springboard for discussing methodological promises and pitfalls of increasingly sophisticated research on the dynamic, contingent, and contextualized features of self-regulated learning. PMID:25825589
Pattern formation of frictional fingers in a gravitational potential

NASA Astrophysics Data System (ADS)

Eriksen, Jon Alm; Toussaint, Renaud; Mâløy, Knut Jørgen; Flekkøy, Eirik; Galland, Olivier; Sandnes, Bjørnar

2018-01-01

Aligned finger structures, with a characteristic width, emerge during the slow drainage of a liquid-granular mixture in a tilted Hele-Shaw cell. A transition from vertical to horizontal alignment of the finger structures is observed as the tilting angle and the granular density are varied. An analytical model is presented, demonstrating that the alignment properties are the result of the competition between fluctuating granular stresses and the hydrostatic pressure. The dynamics is reproduced in simulations. We also show how the system explains patterns observed in nature, created during the early stages of a dike formation.
Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA Polymerase β's activity.

PubMed

Homouz, Dirar; Joyce-Tan, Kwee Hong; Shahir Shamsir, Mohd; Moustafa, Ibrahim M; Idriss, Haitham

2018-01-01

DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity. Copyright © 2017. Published by Elsevier Inc.
Assessing the binding of cholinesterase inhibitors by docking and molecular dynamics studies.

PubMed

Ali, M Rejwan; Sadoqi, Mostafa; Møller, Simon G; Boutajangout, Allal; Mezei, Mihaly

2017-09-01

In this report we assessed by docking and molecular dynamics the binding mechanisms of three FDA-approved Alzheimer drugs, inhibitors of the enzyme acetylcholinesterase (AChE): donepezil, galantamine and rivastigmine. Dockings by the softwares Autodock-Vina, PatchDock and Plant reproduced the docked conformations of the inhibitor-enzyme complexes within 2Å of RMSD of the X-ray structure. Free-energy scores show strong affinity of the inhibitors for the enzyme binding pocket. Three independent Molecular Dynamics simulation runs indicated general stability of donepezil, galantamine and rivastigmine in their respective enzyme binding pocket (also referred to as gorge) as well as the tendency to form hydrogen bonds with the water molecules. The binding of rivastigmine in the Torpedo California AChE binding pocket is interesting as it eventually undergoes carbamylation and breaks apart according to the X-ray structure of the complex. Similarity search in the ZINC database and targeted docking on the gorge region of the AChE enzyme gave new putative inhibitor molecules with high predicted binding affinity, suitable for potential biophysical and biological assessments. Copyright © 2017 Elsevier Inc. All rights reserved.
Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases.

PubMed

Khurana, S; Sanli, G; Powers, D B; Anderson, S; Blaber, M

2000-04-01

2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75. Copyright 2000 Wiley-Liss, Inc.
Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy

PubMed Central

Vallurupalli, Pramodh; Hansen, D. Flemming; Kay, Lewis E.

2008-01-01

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)–ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of 15N, 1HN, 13Cα, and 13CO chemical shifts, along with 1HN-15N, 1Hα-13Cα, and 1HN-13CO residual dipolar couplings and 13CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with 1HN-15N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins. PMID:18701719

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