Application of Distance Learning to Interactive Seminar Instruction in Orthodontic Residency Programs

A series of experiments involving 3 orthodontic departments has shown that distance learning can be acceptable to residents and effective in teaching concepts that are fundamental to orthodontic practice. The improvement from pre- to post-test scores after observing a sequence of distance seminars was similar to direct instruction. Orthodontic residents rated the educational experiences very positively. Live participation in seminars via video conferencing was preferred to live observation or later observation of a recording, but observation provided similar improvement in test scores. The acceptability of the distance seminars appeared to be influenced by the instructor's personality and teaching style in facilitating interaction, the seminar subject, the residents' comfort level in dealing with this technology, and the sequence for interaction vs observation. Further development of recorded seminars with live follow-up discussions has the potential to supplement instruction in graduate orthodontic programs and help with the impending shortage of experienced fulltime orthodontic faculty

Stamina of a non-gasketed flange joint under combined internal pressure, axial and bending loading : an experimental study

The performance of a bolted flange joint is characterized mainly by its 'strength' and 'sealing capability'. A number of numerical and experimental studies have been conducted to study these characteristics under internal pressure loading conditions alone. However, limited work is found in the literature under conditions of combined internal pressure and axial loading. The effect of external, axial loading pressure being unknown, the optimal performance of the bolted flange joint cannot be achieved. Current design codes do not address the effects of axial loading on structural integrity and sealing ability. To study joint strength and sealing capability under combined loading conditions, an extensive experimental and numerical study of a non¬gasketed flange joint was carried out. Actual joint load capacity was determined at both design and test stages with the maximum external axial loading that can be applied for safe joint performance. Experimental and numerical results have been compared and overall joint performance and behaviour is discussed in detail

Computational Study of Protein-Ligand Unbinding for Enzyme Engineering

The computational prediction of unbinding rate constants is presently an emerging topic in drug design. However, the importance of predicting kinetic rates is not restricted to pharmaceutical applications. Many biotechnologically relevant enzymes have their efficiency limited by the binding of the substrates or the release of products. While aiming at improving the ability of our model enzyme haloalkane dehalogenase DhaA to degrade the persistent anthropogenic pollutant 1,2,3-trichloropropane (TCP), the DhaA31 mutant was discovered. This variant had a 32-fold improvement of the catalytic rate toward TCP, but the catalysis became rate-limited by the release of the 2,3-dichloropropan-1-ol (DCP) product from its buried active site. Here we present a computational study to estimate the unbinding rates of the products from DhaA and DhaA31. The metadynamics and adaptive sampling methods were used to predict the relative order of kinetic rates in the different systems, while the absolute values depended significantly on the conditions used (method, force field, and water model). Free energy calculations provided the energetic landscape of the unbinding process. A detailed analysis of the structural and energetic bottlenecks allowed the identification of the residues playing a key role during the release of DCP from DhaA31 via the main access tunnel. Some of these hot-spots could also be identified by the fast CaverDock tool for predicting the transport of ligands through tunnels. Targeting those hot-spots by mutagenesis should improve the unbinding rates of the DCP product and the overall catalytic efficiency with TCP

HotSpot Wizard 3.0: Automated design of site-specific mutations and smart libraries

HotSpot Wizard is an interactive web server for prediction of amino acid residues suitable for mutagenesis and construction of libraries of mutants with modified activity, specificity or stability [1]. Positions suitable for mutagenesis are evaluated based on protein structure using a combination of structural, functional and evolutionary information obtained from 7 internet databases and 22 computational tools [2]. The application was designed with an emphasis on an easy usage without the necessity of advanced knowledge of the studied system. This is the reason for the setting of all default values of the parameters based on the extensive analysis to appropriately represent a wide spectrum of input data. Four different strategies are automatically evaluated for every protein structure. Analysis of the results is being run directly in the web interface, which provides user-friendly visualization tool. Moreover, HotSpot Wizard provides a module for the design of a construction of protein mutant library with the support of an automatic detection of suitable target amino acids and corresponding degenerative codons. There are several new features for the version 3.0 to be released early 2018. Stability of single-point or multiple-point mutant can be predicted using the Rosetta scoring function [3]. Users can newly enter also protein sequence as the input for calculation. Then searching for structures or models in the databases of experimental structures or depositories of homology models is performed. The users can also run homology modelling using the programs Modeller [4] and I-Tasser [5]. The current version of the application is freely available for academic users at http://loschmidt.chemi.muni.cz/hotspotwizard. 1. Pavelka, A., Chovancova, E., Damborsky, J., 2009: HotSpot Wizard: a Web Server for Identification of Hot Spots in Protein Engineering. Nucleic Acids Research 37: W376-W383. 2. Bendl, J., Stourac, J., Sebestova, E., Vavra, O., Musil, M., Brezovsky, J., Damborsky, J., 2016: HotSpot Wizard 2: Automated Design of Site-Specific Mutations and Smart Libraries in Protein Engineering. Nucleic Acids Research 44: W479-W487. 3. Kellogg, E.H., Leaver-Fay, A., Baker, D., 2011: Role of Conformational Sampling in Computing Mutation-induced Changes in Protein Structure and Stability. Proteins 79: 830-838. 4. Sali, A., Blundell, T.L., 1993: Comparative Protein Modelling by Satisfaction of Spatial Restraints. Journal of Molecular Biology 234: 779-815. 5. Zhang, Y., 2008: I-TASSER Server for Protein 3D Structure Prediction. BMC Bioinformatics 9: 40

Comparison of complexity indicators for assessing general process structures

Rad se bavi usporedbom različitih pokazatelja pri procjeni složenosti odabranih općih struktura procesa. Glavni cilj istraživanja je ispitivanje sposobnosti tih pokazatelja u otkrivanju očekivanih razlika u strukturnoj složenosti među promatranim općim strukturama procesa. Rezultati ovog teorijskog istraživanja pokazuju da svi predloženi pokazatelji mogu biti učinkovito upotrijebljeni pri analizi strukturne složenosti općih struktura procesa.This paper focuses on the comparison of different complexity indicators for complexity assessment of selected general process structures. The main objective in this study is to test their ability to uncover assumed differences in structural complexity among observed general process structures. The obtained results of this theoretical study show that all proposed indicators can be effectively used for analyzing structural complexity of general process structures

CaverDock: Software tool for fast screening of un/binding of ligands in protein engineering

Protein tunnels, channels and gates are important for enzymatic catalysis and also represent attractive targets for rational protein design and drug design [1]. Drug molecules blocking the access of natural substrate or release of products are very efficient modulators of biological activity. Here we demonstrate the application of newly in-house developed software tool CaverDock [2,3] for the analysis of the transport of ligands through tunnels in biomolecular targets. Caverdock is a new addition to the Caver Suite [4-6]. We performed virtual screening of large databases of drugs against two pharmacologically relevant targets. We have used FDA-approved drugs for both targets. Oncological drugs (133 molecules), taken from the NIH website, and anti-inflammatory (56 molecules), taken from the Drugbank website, as the libraries of ligands for the two molecular targets: (i) cytochrome P450 17A1 and (ii) leukotriene A4 hydrolase/aminopeptidase. Moreover, we will also show the unbinding of the 2,3-dichloropropan-1-ol product from a buried active site of an haloalkane dehalogenase and its variant. With this study we identified hot-spots that may be used for directed evolution or site-directed mutagenesis to create new variants for faster 2,3-dichloropropan-1-ol release [7]. Finally, we will show the difference on ligand transportation when a protein is in an open and closed conformations [8]. We will show how CaverDock tackles the problem of protein flexibility. 1. Marques, S.M., et al., 2017: Enzyme Tunnels and Gates as Relevant Targets in Drug Design. Medicinal Research Reviews 37: 1095-1139. 2. Vavra, O., et al., 2019: CaverDock 1.0: A New Tool for Analysis of Ligand Binding and Unbinding Based on Molecular Docking. Bioinformatics (under review). 3. Filipovic, J., et al, 2019: A Novel Method for Analysis of Ligand Binding and Unbinding Based on Molecular Docking. Transactions on Computational Biology and Bioinformatics (under review) 4. Chovancova, E., et al., 2012: CAVER 3.0: A Tool for Analysis of Transport Pathways in Dynamic Protein Structures. PLOS Computational Biology 8: e1002708. 5. Jurcik, A., et al., 2018: CAVER Analyst 2.0: Analysis and Visualization of Channels and Tunnels in Protein Structures and Molecular Dynamics Trajectories. Bioinformatics 34: 3586-3588. 6. Stourac, J., et al., 2019: Caver Web 1.0: Identification of Tunnels and Channels in Proteins and Analysis of Ligand Transport. Nucleic Acids Research (under review). 7. Marques, S.M., et al., 2019: Computational Study of Protein-Ligand Unbinding for Enzyme Engineering. Frontiers in Chemistry 6: 650. 8. Kokkonen, P., et al., 2018: Molecular Gating of an Engineered Enzyme Captured in Real Time. Journal of the American Chemical Society 140: 17999–18008

Engineering of haloalkane dehalogenase enantioselectivity towards βbromoalkanes: Open-solvated versus occluded-desolvated active sites

Enzymatic catalysis is widely used for preparing optically pure chemicals. Natural catalysts have to be often optimized to exhibit sufficient enantioselectivity towards industrially attractive non-natural substrates. Understanding the molecular basis of enzyme–substrate interactions involved in enantiodiscrimination is essential for rational design of selective catalysts. Haloalkane dehalogenases (EC 3.8.1.5) can convert a broad range of halogenated aliphatic compounds to their corresponding alcohols via SN2 mechanism [1]. The very first haloalkane dehalogenase exhibiting high enantioselectivity towards β-brominated alkanes (E-values of up to 174) was DbjA from Bradyrhizobium japonicum USDA110 [2]. This enzyme has a wide open solvent-accessible active site and its enantioselectivity towards β-brominated alkanes is modulated by a surface loop unique to DbjA [2]. Assuming that the active site geometry is crucial for substrate recognition, it was proposed that DbjA’s enantioselectivity could be transferred to closely related, but non-selective DhaA from Rhodococcus rhodochrous NCIMB13064 [1] by active site transplantation [3]. The unique loop fragment from DbjA together with additional 8-point substitutions was inserted to DhaA. Although the crystal structure of resulting variant DhaA12 exhibited identical geometry of the active site and the access tunnel as DbjA, it did not reach identical level of hydration and flexibility and lacked enantioselectivity towards β-bromoalkanes (E-value = 18) [3]. Interestingly, the variant DhaA31 constructed independently with a goal to enhance enzyme activity towards anthropogenic compound 1,2,3-trichlopropane [4], exhibited high enantioselectivity towards 2-bromopentane (E-value = 179) [5] as DbjA (E-value = 174) [2, 3]. DhaA31 contains five mutations, I135F, C176Y, V245F, L246I and Y273F, located in a main and a slot tunnel. Four of five mutations are large and aromatic residues narrowing two access tunnels and occluding the enzyme active site [4]. The level of DhaA31 active site hydration, so important for DbjA’s enantioselectivity [2, 3] is low, suggesting a different structural basis of enantioselectivity towards 2-bromopentane. A systematic study on the molecular basis of enantioselectivity in DbjA, DhaA, and DhaA31 using thermodynamic and kinetic analyses, site-directed mutagenesis, and molecular modeling was carried out. DhaA31 enantioselectivity arises from the hydrophobic substrate’s interactions with the occluded and desolvated active site [5], while DbjA enantioselectivity results from water-mediated interactions of 2-bromopentane with the active site’s hydrophobic wall [2]. Our data imply that enantioselectivity of haloalkane dehalogenases can be achieved by both occluded-desolvated active site and open-solvated active site. The engineering of “DbjA-like” enantioselectivity by modification of the active site hydration remains challenging. References: 1. Koudelakova, T., et al. 2013. Biotechnol. J. 8: 32–45. Prokop, Z., et al. 2010. Angew. Chem. Int. Ed., 49: 6111-6115. Sykora, J., et al. 2014. Nat. Chem. Biol., 10: 428-430. Pavlova, M., et al. 2009. Nat. Chem. Biol., 5: 727-733. Liskova, V., et al. 2017. Angew. Chem. Int. Ed., DOI: 10.1002/anie.201611193

FireProt ASR: Automated design of ancestral proteins

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EnzymeMiner: Exploration of sequence space of enzymes

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FireProt: Web server for automated design of thermostable proteins

Stable proteins are used in numerous biomedical and biotechnological applications. Unfortunately, naturally occurring proteins cannot usually withstand the harsh industrial environment, since they are mostly evolved to function at mild conditions. Therefore, there is a continuous interest in increasing protein stability to enhance their industrial potential. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. A much higher degree of stabilization can be achieved by the construction of the multiple-point mutants. Here, we present the FireProt method [1] and the web server [2] for the automated design of multiple-point mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen bioinformatics tools, including several force field calculations. Highly reliable designs of the thermostable proteins are constructed by two distinct protein engineering strategies, based on the energy and evolution approaches and the multiple-point mutants are checked for the potentially antagonistic effects in the designed protein structure. Furthermore, time demands of the FireProt method are radically decreased by the utilization of the smart knowledge-based filters, protocol optimization, and effective parallelization. The server is complemented with an interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable proteins. The server is freely available at http://loschmidt.chemi.muni.cz/fireprot. 1. Bednar, D., Beerens, K., Sebestova, E., Bendl, J., Khare, S., Chaloupkova, R., Prokop, Z., Brezovsky, J., Baker, D., Damborsky, J., 2015: FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants. PLOS Computational Biology 11: e1004556. 2. Musil, M., Stourac, J., Bendl, J., Brezovsky, J., Prokop, Z., Zendulka, J., Martinek, T., Bednar, D., Damborsky, J., 2017, FireProt: Web Server for Automated Design of Thermostable Proteins, Nucleic Acids Research, in press, doi: 10.1093/nar/gkx285