Engineering enzyme access tunnels

Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes

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

Caver Web 1.0: identification of tunnels and channels in proteins and analysis of ligand transport

Caver Web 1.0 is a web server for comprehensive analysis of protein tunnels and channels, and study of the ligands’ transport through these transport pathways. Caver Web is the first interactive tool allowing both the analyses within a single graphical user interface. The server is built on top of the abundantly used tunnel detection tool Caver 3.02 and CaverDock 1.0 enabling the study of the ligand transport. The program is easy-to-use as the only required inputs are a protein structure for a tunnel identification and a list of ligands for the transport analysis. The automated guidance procedures assist the users to set up the calculation in a way to obtain biologically relevant results. The identified tunnels, their properties, energy profiles and trajectories for ligands’ passages can be calculated and visualized. The tool is very fast (2–20 min per job) and is applicable even for virtual screening purposes. Its simple setup and comprehensive graphical user interface make the tool accessible for a broad scientific community. The server is freely available at https://loschmidt.chemi.muni.cz/caverweb.Caver Web 1.0 je webový server pro komplexní analýzu tunelů a kanálů v proteinech a pro studium transportu ligandu přes tyto transportní cesty. Caver Web je první interaktivní nástroj umožňující obě analýzy v jednom grafickém uživatelském rozhraní. Server je vybudován nad hojně užívaným nástrojem pro detekci tunelů Caver 3.02 a nad CaverDock 1.0 umožňujícím studium transportu ligandů. Program se snadno ovládá, jelikož vyžaduje pouze strukturu proteinu pro identifikaci tunelů a seznam ligandů pro analýzu transportu. Procedury pro automatické nastavení výpočtů asistují uživatelům tak, aby získali biologicky relevantní výsledky. Identifikované tunely, jejich vlastnosti, energetické profily a trajektorie průchodů ligandů mohou být spočítány a vizualizovány. Nástroj je velmi rychlý (2-20 minut na úlohu) a je použitelný dokonce pro virtuální screening. Jeho snadné nastavení a ucelené grafické rozhraní dělá nástroj přístupným pro širokou vědeckou komunitu. Server je volně k dispozici na https://loschmidt.chemi.muni.cz/caverweb

Dietary supplement of conjugated linoleic acids or polyunsaturated fatty acids suppressed the mobilization of body fat reserves in dairy cows at early lactation through different pathways

To investigate the metabolic (.11, !I:2.es in the adipose tissue (AT) of dairy cows under milk fat depression (MFD), 30 cows were randomly allocated to a control diet, a conjugated linoleic acid (CLA)-supplemented diet, or a high-starch diet supplemented with a mixture of sunflower and fish oil (2:1; as HSO diet) from 1 to 112 d in milk. Performance of animals, milk yield, milk composition, energy balance, and blood metabolites were measured during lactation. Quantitative PCR analyses were conducted on the AT samples collected at wk 3 and 15 of lactation. The CLA and HSO diets considerably depressed milk fat yield and milk fat content at both wk 3 and 15 in the absence of significant changes in milk protein and lactose contents. In addition, the HSO diet lowered milk yield at wk 15 and decreased dry matter intake of cows from wk 3 to 15. Compared with the control, both CLA and HSO groups showed reduced body weight loss, improved energy balance, and decreased plasma concentrations of nonesterified fatty acids and beta-hydroxybutyrate at early lactation. The gene expression analyses reflected suppressed lipolysis in AT of the CLA and HSO groups compared with the control at wk 3, as suggested by the downregulation of hormone-sensitive lipase and fatty acid binding protein 4 and the upregulation of perilipin 2. In addition, the HSO diet promoted lipogenesis in AT at wk 15 through the upregulation of 1-acylglycerol-3-phosphate O-acyltransferase 2, mitochondria' glycerol-3-phosphate acyltransferase, perilipin 2, and peroxisome proliferator-activated receptor gamma. The CLA diet likely regulated insulin sensitivity in AT as it upregulated the transcription of various genes involved in insulin signaling, inflammatory responses, and ceramide metabolism, including protein kinase B2, nuclear factor kappa B1, toll-like receptor 4, caveolin 1, serine palmitoyltransferase long chain base subunit 1, and N-acylsphingosine amidohydrolase 1. In contrast, the HSO diet resulted in little or no change in the pathways relevant to insulin sensitivity. In conclusion, the CLA and HSO diets induced a shift in energy partitioning toward AT instead of mammary gland during lactation through the regulation of different pathways.Peer reviewe

Strategies and software tools for engineering protein tunnels and dynamical gates

Improvements in the catalytic activity, substrate specificity or enantioselectivity of enzymes are traditionally achieved by modification of enzymes’ active sites. We have recently proposed that the enzyme engineering endeavors should target both the active sites and the access tunnels/channels [1,2]. Using the model enzymes haloalkane dehalogenases, we have demonstrated that engineering of access tunnels provides enzymes with significantly improved catalytic properties [3] and stability [4]. User-friendly software tools Caver [5], Caver Analyst [6], CaverDock [7] and Caver Web [8], have been developed for the computational design of protein tunnels/channels; FireProt [9] and HotSpot Wizard [10] for automated design of stabilizing mutations and smart libraries. Using these tools we were able to introduce a new tunnel to a protein structure and tweak its conformational dynamics. This engineering strategy has led to improved catalytic efficiency [2], enhanced promiscuity or even a functional switch (unpublished). Our concepts and software tools are widely applicable to various enzymes with known structures and buried active sites. 1. Damborsky, J., et al., 2009: Computational Tools for Designing and Engineering Biocatalysts. Current Opinion in Chemical Biology 13: 26-34. 2. Prokop, Z., et al., 2012: Engineering of Protein Tunnels: Keyhole-lock-key Model for Catalysis by the Enzymes with Buried Active Sites. Protein Engineering Handbook, Wiley-VCH, Weinheim, pp. 421-464. 3. Brezovsky, J., et al., 2016: Engineering a de Novo Transport Tunnel. ACS Catalysis 6: 7597-7610. 4. Koudelakova, T., et al., 2013: Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel. Angewandte Chemie 52: 1959-1963. 5. Chovancova, E., et al., 2012: CAVER 3.0: A Tool for Analysis of Transport Pathways in Dynamic Protein Structures. PLOS Computational Biology 8: e1002708. 6. 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. 7. 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). 8. 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). 9. Musil, M., et al., 2017: FireProt: Web Server for Automated Design of Thermostable Proteins. Nucleic Acids Research 45: W393-W399. 10. Sumbalova, L. et al., 2018: HotSpot Wizard 3.0: Automated Design of Site-Specific Mutations and Smart Libraries in Protein Engineering. Nucleic Acids Research 46: W356-W362

Intranet ja sosiaalinen media organisaation sisäisessä viestinnässä

The purpose of the thesis was to study how the client organisation’s new intranet functions in work community communication and in internal communication The aim was also to find out if the employees use the tools of social media on the intranet. The thesis is built on the notion that intranet improves organisations internal communication and utilizing an intranet widely helps to helps to enhance communication among the employees and creates new interactive communication possibilities. The thesis sought answers to the following questions: How is an intranet perceived as a tool of internal communication? What kind of information do employees search on the intranet and does the content meet their needs? How widely are social media tools and practices used on an intranet? Are the employees ready to use these tools and do they see them useful? The theory part of the thesis explored general scientific theories and literature on social media jne. In the research part of the thesis a quantitative survey was made among the personnel of the client organisation. The last part of the thesis contains the analysis of the responses in the survey, conclusions and proposals for action. The study demonstrated that an intranet is a significant tool for internal communication. It is widely used in the client organisation. On the contrary, social media tools are not so often used on the client organisation's intranet. The employees do not consider them useful; they are also less aware of the tools.Opinnäytetyön tarkoituksena oli selvittää toimeksiantajaorganisaation uuden intranetin toimivuutta sisäisen viestinnän välineenä sekä kuinka intranetissä olevat sosiaalisen median työkalut on kohde-organisaatiossa otettu käyttöön. Opinnäytetyössä etsittiin vastausta intranetin merkitykselle sisäisen viestinnän välineenä sekä tutkittiin onko intranetissä tarvetta sosiaalisen median työkaluille ja aut-tavatko ne vuorovaikutteisuuden ja avoimuuden kulttuurin lisäämisessä. Opinnäytetyön johtoajatuksena oli, että intranetin avulla voidaan parantaa organisaation sisäistä viestintää ja intranetin monipuolinen hyödyntäminen organisaatiossa auttaa parantamaan organi-saation työntekijöiden keskinäistä kommunikointia ja luomaan uusia vuorovaikutteisia viestintämah-dollisuuksia. Opinnäytetyön toteuttaminen rakentui teoriaosaan, jossa perehdyttiin kirjallisuuteen, artikkeleihin ja muihin lähteisiin koskien sisäistä viestintää ja sosiaalisen median työkaluja. Teoriaperustaa seu-rasi tutkimusosuus, jossa toteutettiin tutkimus kvantitatiivisena kyselynä koko organisaation henki-lökunnalle. Viimeisessä osiossa analysoitiin tutkimuksen tulokset ja esitettiin niihin perustuvat joh-topäätökset ja jatkotoimenpide-ehdotukset. Kysely osoitti, että intranetillä on suuri merkitys kohdeorganisaatiossa tiedon lähteenä sekä sisäi-sen viestinnän välineenä. Intranetistä etsitään eniten tietoa ajankohtaisista asioista, tietoa koulu-tuksista sekä ohjeita ja lomakkeita. Uusi intranet on otettu hyvin käyttöön, mutta siinä koetaan olevan vielä puutteellisuuksia tietojen löydettävyydessä. Intranetissä olevat sosiaalisen median työkalut sen sijaan on otettu aika vähäisessä määrin käyttöön ja eikä niitä koeta kovinkaan hyödyl-lisinä. Kohdeorganisaation intranet on toimiva sisäisen viestinnän väline ja intranetin sisältö vastaa hyvin työntekijöiden tarpeisiin. Sisältöä tulee kuitenkin kehittää ja muistaa pitää se ajantasaisena käyttö-aktiivisuuden ylläpitämiseksi. Sosiaalisen median työkalujen mahdollisuuksia tulee tuoda enemmän esille ja yrittää saada niihin hyödyllisiä ja mielenkiintoisia sisältöjä käytön lisäämiseksi

Stormflow against streamflow – Can LID-provided storage capacity ensure performance efficiency and maintenance of pre-development flow regime?

Funding Information: This work was funded by Ministry of Agriculture and Forestry, Finland; Schlumberger Foundation Faculty for the Future, Academy of Finland (no 326787, WaterWorks2017 ERA-NET Cofund) and Maa- ja vesitekniikan Tuki ry. The study was part of the UrbanStormwaterRisk and EviBAN (Evidence based assessment of NWRM for sustainable water management) projects. Elisa Lähde, a landscape architect, designed the alternative stormwater management designs assessed in this study. The rainfall data for design simulations came from a rain gauge operated by the City of Turku. The streamflow data for rural reference watershed (Savijoki) and semi-urban reference watershed (Kuninkoja) came from monitoring stations maintained by the Finnish Environment Institute (SYKE) and Turku University of Applied Sciences (TUAS), respectively. Funding Information: This work was funded by Ministry of Agriculture and Forestry, Finland; Schlumberger Foundation Faculty for the Future, Academy of Finland (no 326787, WaterWorks2017 ERA-NET Cofund) and Maa- ja vesitekniikan Tuki ry. The study was part of the UrbanStormwaterRisk and EviBAN (Evidence based assessment of NWRM for sustainable water management) projects. Elisa Lähde, a landscape architect, designed the alternative stormwater management designs assessed in this study. The rainfall data for design simulations came from a rain gauge operated by the City of Turku. The streamflow data for rural reference watershed (Savijoki) and semi-urban reference watershed (Kuninkoja) came from monitoring stations maintained by the Finnish Environment Institute (SYKE) and Turku University of Applied Sciences (TUAS), respectively. Publisher Copyright: © 2021 The Author(s)The goal of Low Impact Development (LID) is to restore and maintain the pre-development flow regime. The static storage capacity, which is often used as a parameter in LID designs, provides the maximum capacity of an LID type and is easily quantifiable already at the design phase. However, the static storage approach does not consider the inter-event recovery of storage capacity by infiltration and evapotranspiration. This study investigated dynamic storage capacities of three stormwater management designs with increasing proportions of LID units on a 1.2 ha urban residential block in Southern Finland, to compare their cost-efficiency, as well as their potential in restoring the pre-development flow regime. The cost-efficiency of LID designs was assessed based on their ability to contribute to water losses, and on the additional construction costs required when comparing them to conventional solutions (e.g. asphalt replaced with permeable pavement). The design with a small storage capacity and a large captureratio, i.e., the ratio of contributing area to LID area, was the least efficient albeit its small construction cost. The design with an appropriate balance between the capture ratio and the LID provided storage capacity was the most efficient option. In assessing the potential of stormwater designs in restoring the pre-development flow regime, the sum of infiltration and flow in storm sewer networks was more representative of the catchment total runoff than flow alone. Finally, an extensive simulation of a large set of differently placed LID units proved useful in a priori identification of the most influential units in the treatment train.Peer reviewe

Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

The ability to engineer enzymes for industrial and biomedical applications is primarily limited by a paucity of mechanistic understanding. To gain insight into the mechanisms of enzyme catalysis, one must screen enormous numbers of discrete reaction conditions, which is a laborious task using conventional technologies. To address such limitations, we develop a droplet-based microfluidic platform for high-throughput acquisition of transient kinetic data over a range of substrate concentrations and temperatures. When compared with conventional methods, our platform reduces assay volumes by six orders of magnitude and increases throughput to 9,000 reactions/min. To demonstrate their utility, we measure the transient kinetics of three model enzymes, namely, β-galactosidase, horseradish peroxidase, and microperoxidase. Additionally, we conduct a complex kinetic and thermodynamic study of engineered variants of haloalkane dehalogenases. Datasets are globally analyzed and complemented by molecular dynamics simulations, providing new insights into the molecular basis of substrate specificity and the role of hydration-related entropy.ISSN:2451-9294ISSN:2451-930