• 10th anniversary

  • June 2024 - cyanobacterial DD-peptidase story published

  • February 2024 - reducing drought stress story published

  • June 2023 - TLRs dedicated review.

  • April 2023 We are organizing international workshop on proteins ligands interaction.

  • February 2023 New publication in Plos ONE

  • November 2022 - new publication in JCIM.

  • May 2022 New Publication in Plos Computational Biology


We are using an advanced theoretical approach to investigate the properties of various enzymes in order to design biologically active compounds and to help experimentalists interpret their results.

The Tunneling Group was established in June 2014 as an independent research group.

The main activity of the group lies on the border of molecular biology and computational chemistry. We are using an advanced theoretical approach to investigate properties of various enzymes, design biologically active compounds, and help experimentalists in interpretation of their results.

We are employing molecular dynamic simulations, various bioinformatics tools and in-house know-how methods to improve selectivity, activity and stability of enzymes and to explore structure-function relationship of proteins. Instead of modifying active site residues, we are exploring the role of distant residues which can tune properties of biocatalysts and optimise their performance. Our rational approach explores multidimensional space of conformational changes, provides a deep understanding of catalysis events, and results in the design of smart libraries of enzyme mutants.

We are using knowledge of structural-function relationship as a starting point for optimisation of known active compounds, searching for new ones (extensive screening for chemicals which might be used as inhibitors or activators of macromolecules involved in medical or industrial applications), and identification of side targets causing toxicity. We have well-established collaboration with national and international partners from academia, health, and industry.

The experimental part of the Tunneling Group is focused on production and optimization of proteins and bioactive compounds, and on biochemical and biophysical characterisation of enzymes and macromolecules. We have our own high-throughput facility for protein production, purification, binding affinity measurement and activity testing.

Our research covers three interpenetrating areas including biochemistry and catalysis, health, and environment sectors. Our interests are focused on basic and applicable studies including optimisation of enzymes, improvement of their activity and selectivity, development of new drugs (against COVID-19, cancer, cardiovascular and neurodegenerative diseases), antimicrobial peptides and inhibitors (against forest pests) and insight into molecular mechanism standing behind rare diseases and catalysis.

We are located in the Biotechnology Centre, which was established in the Silesian University of Technology (SUT) in 2006. The Biotechnology Centre is also one of the partners of the BIO-FARMA consortium formed in April 2007 by the SUT, National Research Institute of Oncology, Silesian Medical University and the University of Silesia – thus it opens easy access to various collaboration and interdisciplinary projects.


Gamze was representing our group on the First Minisymposium on ...
Sincerely congratulations to Maria Bzówka for outstanding defense of her ...
Agata Raczyńska, Katarzyna Szleper and Artur Góra were representing our ...
Kasia and Maria were presenting their study " Studying the ...
Gamze Tanriver was selected for Flagship Workshop organized by CECAM ...
Maria will present her work during EMBO Lecture Course "Structural ...
We were selected as one of the presenters during Silesian ...
From 2024 our group will grow again. We will have ...
We will have lecture during Scientific nights 7 October 2023 ...
Great news - Oksana has received prestigious EMBO Solidarity grant ...
Together with NanoTemper Technologies we are organizing Symposium & Workshop ...
Great news from Lisbon! Maria is representing our group during ...
Biochemists and protein chemists MD Sarfaraz Alam and computational chemists ...
Our team member Weronika Bagrowska was awarded by the Ministry ...
"Searching for novel potential binding sites and small-molecule inhibitors towards ...
Manuscript "Structural analysis of the effect of Asn107Ser mutation on ...
We are offering support for scientists and students from Ukraine ...
Sincerely congratulations to Jakub. His case report presented during the ...
Jakub Mróz's presentation was qualified to contest session during students ...
Maria Bzówka application on YSF 2022 and the ensuing IUBMB–FEBS–PABMB ...
Our new work about potential inhibitors of the SARS-CoV-2 Mpro ...
The results of pro-quality competition for rector's grants for highly ...
Maria Bzówka was awarded by HPC-Europa3 Programme grant and she ...
Sincerely congratulations to Weronika. Her engineering thesis about FUT8 inhibitors ...
Jaspreet Jandoo has joined our team and will work with ...
Our communicate about potential group of compounds that can target ...
Jakub Mróz has joined our team. He will strength our ...
Our work published in Drug Discovery Today has initiated collaboration ...
We are extremely happy!! Our proposal (with NMBU, TU-Graz and ...
Marzena has joined Doctoral School at Sileian University of Technology ...
Khadija Batool will join our team and will work as ...
Maria is starting her 1 month stay in Horacio Perez ...
Our group leader is starting 3 months research stay at ...
Agata is starting her nearly 4 months stay in Isabelle ...
We are looking for postdoctoral researcher to conduct interdisciplinary research ...
Our 4 years project dedicated to the development of safe ...
Our joint publication with authors from the Basel University - ...
Divine’s project was awarded in Miniature 4 call from National ...
Katarzyna’s and Divine’s projects awarded in the pro-quality programme from ...
Our research work presented during the first edition of the ...
Our project awarded in the 4th competition within the framework ...
Our research will be supported by the Plastic Omnium Auto ...
Welcome new members - Katarzyna and Marek! ...
2020-09-23 – First edition of AQUA-DUCT webinar was organized online ...
Maria was awarded with the scholarship for the best PhD ...
Congratulations! ...
Agata awarded in 9th edition of the Diamond Grant competition ...
Agata was awarded with the French Government scholarship for doctorate ...
Patryk's Master Thesis was awarded with distinction by the Faculty ...
Congratulations to Divine for successful nostrification of his PhD in ...
Our publications awarded in the pro-quality programme from the Silesian ...
Our publication about SARS-CoV2 Mpro has been just published. Open ...
New publication from our group has been just published: Simple ...
New publication from our group has been just published: Structure ...
The school will provide an opportunity to learn basic and ...
We are opening a new laboratory in Grey Chemistry building ...
Congratulations to Agata. She was awarded by prestigious scholarship from ...
Publication describing new version of the AQUA-DUCT tool is available ...
Congratulations to Maria and Agata. Maria got the Omnium Studiosorum ...
The Monolith NT.Automated system - the high-throughput system for ligands ...
The new version of AQUA-DUCT is combining information from small ...
Does the loop reconstruction method affect protein functionality? The poster ...
Our team member was selected as a one of the ...
Our last publication has been selected as a cover story ...
New publication in cooperation with groups from UK and The ...
New technologies and approaches for human diagnostics and life science ...
Agnieszka has published her work on Reaction mechanism between Cu(II)-enolate ...
The School on molecular computational biochemistry has placed in Krakow, ...
Congratulations to Maria and Agata. They were awarded by prestigious ...
It was a great and intensive time in Zwardoń. Thank ...
The 5th edition of the Projects Days will take place ...
The overview of AQUA-DUCT software was presented at School of ...
The Workshop on Biorefinery Enzymes for Renewable Chemicals: From Discovery ...
Our work concerning analysis of important futures of the
epoxide ...
Our work will be presented during 'Enzymes, biocatalysis and chemical ...
An AQUA-DUCT will be presented during Application Track session at ...
Our new tool an R package for entropy/variability analysis was ...
Evolutionary analysis of epoxide hydrolases, water as a molecular probe ...
The EMBO Practical Course on Integrative modelling of biomolecular interactions ...
You can see our unpublished work during Faraday Discussion on ...
"AQUA-DUCT: a solvent and ligands tracking tool" has been accepted ...
Our work concerning modification of the D-amino acids oxidase was ...
Maria application was accepted for EMBO Practical Course Integrative modelling ...
Kasia is participating in FEBS course - FEBS Integrated Approaches ...
Welcome new team members - Weronika, Aleksandra and Mariusz on ...
We will organize a workshop for AQUA-DUCT users during 10th ...
Petra and Klaudia from Loshmidt Laboratories are visiting our group ...
Agnieszka enginnering project has been selected by Faculty of Chemistry ...


June 2023



Karolina Mitusińska - Google Scholar - recent location

Tomasz Magdziarz - Google Scholar

Agnieszka Stańczak

Piotr Wojsa

Aleksandra Samol

Sandra Gołdowska

Magdalena Ługowska

Mariusz Wiecha

Alicja Płuciennik

Michał Stolarczyk

Patrycja Spychalska

Patryk Kapica

Agata Hadryś

Klaudia Strama

Natalia Radlak

Karolina Leszczyńska

Agnieszka Pietka

Mateusz Tomczyk

Mariusz Nowak

Mateusz Kuc

Anna Janowska

Angelika Karasewicz

Maciej Opałka

Ongoing Projects

Teaching Lytic Polysaccharide Monooxygenases to do Cytochrome P450 Catalysis

“Lytic polysaccharide monooxygenases (LPMO) and cytochrome P450 (CYP) are copper- and iron-dependent, respectively, enzymatic systems that perform regio- and stereospecific oxidation of non-activated hydrocarbons in Nature. To control such reactions in modern industry and biotechnology is of utmost importance in creating products of value such as secondgeneration bioethanol and products of value for i.e. the pharmaceutical industry. Due to the major drawbacks of using CYPs, including their partially membrane bound nature and the requirement of a reductase in combination with reducing agents such as NAD(P)H to transfer electrons to the active site for oxygen activation, it is highly desirable to develop new type of catalyst that can perform the same type of reactions. An attractive alternative strategy is to engineer LPMOs to perform CYP catalysis. LPMOs are small, robust, easy to produce in large scale, and rigid water-soluble proteins with a plethora of electron donors. The extended, flat LPMO surface, with huge natural sequence variation and thus, likely, mutability, provides a fantastic scaffold for engineering access to the active site as well as substrate affinity. We propose to use LPMOs engineered to accommodate typical CYP substrates and immobilize this on solid supports to provide confinement necessary in bringing the oxygen species together with the C-H bond to be oxidized in a tailored, “”closed”” environment. Moreover, the rate of LPMO catalysis can be greatly enhanced compared to traditional CYP catalysis by the addition H2O2 in the presence of low, priming concentrations of an external reductant to achieve efficiency constants (kcat/Km) in the order of 106 M-1s-1, which is typical for peroxygenases. The proposed ground-breaking research fits excellently well with the work program “”Future and Emerging Technologies”” where the goal is to challenge current thinking.”

In cooperation with:

Project is financed by:

Combining specialists from chemistry, biology, microbiology, biotechnology, entomology and ecology to search for safe and selective pesticides. 

Since the dawn of time, humanity encountered a great problem with agricultural pests – viruses, bacteria, undesirable fungi, plants, and animals. The largest group of pests are insects, affecting not only agriculture but also forest areas. Insects can be vectors of dangerous diseases and negatively affect the health of plants, animals, and humans. On the other hand, they are an extremely important part of the ecosystem. For example, approximately 75% of crop species in agricultural and horticultural cropping systems are pollinated by insects. Therefore, pesticides and insecticides has to be designed with extreme care.

Extensive research on insect physiology has provided a lot of information about their  anatomy, organs, structures, and functioning. Great amount of work were devoted to analysing the insects development process. It was found, that the juvenile hormone (JH) is crucial in the insects’ metamorphosis. Nowadays, it is thought, that the juvenile hormone epoxide hydrolase is a key enzyme in this process. The inhibition of JHEH could prevent the development of an adult form of insects and consequently lead to the degradation of particular species. It would be desirable in the case of insects that are agricultural and forest pests, but also in relation to insects, which can be vectors of dangerous diseases.

The proposed project brings together different fields of research: chemistry, biology, microbiology, biotechnology, entomology and ecology. In the proposed project, we would like to use in silico methods to identify differences in the structural properties and dynamics of JHEH among different insect of closely related species. By carrying out a comprehensive structural analysis focused mainly on the definition and description of intramolecular voids that are available for the binding of small molecules, it will be possible to identify regions in JHEH that are unique to individual insect species and potentially able to distinguish selective inhibitors dedicated to specific pests and safe to other insects, plants and animals. The proposed compounds will be tested also for environmental safety.

Project is financed by:

The bacterial peptides with antimicrobial activity, called bacteriocins can be used as an environment-friendly and what is the most important efficient alternative to antibiotics.

This project aims to identify, characterize and demonstrate the antimicrobial activity of bacteriocins derived from the marine environment. There is an increasing concern about antibiotics crisis in recent years due to the unconscious and extensive use of antibiotics, which causes resistant bacteria to evolve and decreases the effectiveness of the current selection of antibiotics available. In this project, we aim to explore and exploit the potential of Turkish marine habitats in terms of antimicrobials that can be used as an environment-friendly and what is the most important efficient alternative to antibiotics. The proposed project aims to identify bacterial peptides with antimicrobial activity, called bacteriocins, from the marine environment, characterize selected bacteriocins, test their antimicrobial activity against various human pathogens, investigate the synergistic activity of bacteriocins with antibiotics and determine bacteriocins’ environmental impacts falls within the scope of the call. Previous studies were mainly focused on bacteriocins produced by terrestrial species. Even though there are marine bacteriocin studies conducted, they usually did not provide an in-depth characterization. They are however very important and promising in the medical treatment, since they can persist in highly saline habitats. This study aims to benefit from a unique source, Sea of Marmara, which is subjected to high levels of anthropogenic pressures due to its proximity to the most populated city of Europe, Istanbul and small exchange of water with surrounding basins. Both sediment and water samples will be collected from Istanbul shores, Izmit Bay and Kocaeli Municipal Wastewater Treatment Plant, which were found to be rich in multiple drug-resistant bacteria. The expected outcomes of this project will be obtaining antibiotic alternatives from marine environments with complex microbial community and impacted by human activity, in-depth characterization of isolated bacteriocins’ innate and synergistic activity against human pathogens and revealing their potential to be used as a treatment/supplement that is safer for the environment. The main research activities will be (i) identification and isolation of bacteriocin genes (ii) expression and characterization of selected bacteriocins (iii) testing microbial activity of bacteriocins against human pathogens (iv) modeling and insight into structural characterization of most potent bacteriocins and (v) evaluation of the risk of the off-target activity.

In cooperation with:

Project is financed by:

Enzymes can be a great weapon in our fight with wastes. Here we will implement stat-of-the-art in silico approaches to re-engineer natural catalysts for polyurethane decomposition. 

Plastics formed by synthetic polymers are durable materials that possess many desirable features, but their high resistance to biodegradability, once considered an advantage, is now one of the main causes of environmental problems in the world. Every year, due to inadequate recycling of plastics, millions of tons of waste accumulate in terrestrial and aquatic environments, exerting a damaging effect on them. The micro and nanoparticles of plastics, formed as a result of physical erosion processes, pose a new and previously unknown threat to human and animal health. Effective methods of degradation of these synthetic polymers are needed. An alternative and environmentally friendly method of degradation of plastics is enzymatic biocatalysis. It is known that some microorganisms can colonize the surface of plastics and are able to slowly degrade it, however, this applies only to selected plastics, and the enzymes and mechanisms of the process of hydrolysis and/or oxidation of synthetic polymers are not well understood.

The aim of the proposed project is to find and learn about enzymes capable of binding short oligomers of synthetic polymers. Synthetic polymers, that were selected as subjects of this research are commonly used plastics: polyurethane (PUR), polystyrene (PS) and polyethylene (PE). The gained knowledge will be used to redesign the selected enzymes to increase its stability, affinity and activity, and if it is possible to expand its substrate selectivity.

Modern in silico techniques will be applied in the project, which will include molecular dynamics simulations, molecular docking, protein-ligand interaction analysis and computational enzyme design. It is assumed that as a result of the project, using in silico techniques, it will be possible to learn about the molecular aspects responsible for recognizing and binding polymer chains. Identification of key amino acids in this process will allow to optimize the surface of the protein responsible for recognizing
the substrate in order to increase the activity and modify the substrate specificity of enzymes capable of degrading synthetic polymers. The results of the computational work will also be experimentally verified.

Project is financed by:

How to inhibit the main protease of the SARS-CoV2 virus? We are using combination of the molecules tracking and local distribution approach to help answer that question.

To this date, we performed over 2µs of classical molecular dynamics simulations (cMD) of both SARS and SARS-CoV-2 main proteases (Mpros) as well as nearly 1 µs of mixed-solvents molecular dynamics simulations (MixMD) with various cosolvents including acetonitrile, benzene, dimethylsulfoxide, methanol, phenol, and urea [1]. The combined small molecules’ tracking approach and local distribution analysis were used to analyse the conformational changes in the binding site, as well as to detect other potential binding sites that could be used for inhibitors design. Our results indicate, that there are large differences between plasticity and size of the SARS-CoV and SARS-CoV-2 Mpro protease and moreover, the access to the active site can be regulated by the C44-P52 loop which is carrying one of the unique for SARS-CoV-2 Mpro amino acid [1].

Additionally, the preliminary analysis of the potential mutability of the active site surrounding was performed. The single nucleotide substitutions were introduced to the SARS-CoV-2 main protease gene and their energetic contributions to protein stability were calculated. The most important message comes from the analysis of the potential mutability of the C44-P52 loop. The mutation of four of them has a stabilising effect for the protein and for the rest the effect is near-neutral. These results indicate that the future evolution of the Mpro protein in this region is likely to occur and can significantly reduce the potential use of the drugs developed at this stage of research due to a highly probable development of drug resistance of this virus through mutations. Therefore our study is of significant importance and can provide a contribution to the development of future therapeutic strategies.

The preliminary results. (A) Comparison of cosolvent distribution in the active site of SARS-CoV-2 and SARS-CoV Mpro proteins during MixMD simulations. (B) Predicted stability of the residues of the active site cavity. The green colour indicate residues prone to mutate.
  1. Papaj K., et al. Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease. PloS ONE 2022, 17 (1), e0262482 doi: 10.1371/journal.pone.0262482 ARTICLEOpen Access
  2. Papaj K., et al. Investigation of Thiocarbamates as Potential Inhibitors of the SARS-CoV-2 Mpro Pharmaceuticals 202114(11), 1153 doi: 10.3390/ph14111153 ARTICLEOpen Access
  3. Fischer A., et al. Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2 Int. J. Mol. Sci. 2021, 22(4), 2065 doi: 10.3390/ ijms22042065 ARTICLEOpen Access
  4. Bzówka M., et al. Structural and Evolutionary Analysis Indicate That the SARS-CoV-2 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design, Int. J. Mol. Sci. 2020, 21(7), 3099 doi: 10.3390/ijms21093099 ARTICLEOpen Access

TLRs are large and diverse transmembrane proteins that are essential for signal transduction. We aim to study how important are water molecules in molecular response mediation.

Water molecules take part in metabolic reactions, they constitute an intracellular transport environment but also are involved in dynamic changes in protein structures, including transmembrane proteins. It is assumed that they can affect signal recognition, as well as conformational changes caused by interaction with ligands or adapter proteins, or the process of signal transmission across the cell membrane. 

In most of the known studies, the analysis of water behaviour in intermolecular interactions has been omitted. Therefore, this project aims to determine whether water molecules can actually act as a mediator in protein regulation. The biological system which fits well for this study is the family of transmembrane Toll-like receptors (TLRs). These receptors recognise and interact with a wide spectrum of compounds – from low molecular compounds, through nucleic acids, peptides, to whole proteins. TLRs are a class of proteins that play a key role in the innate immune system. 

The proper proteins regulation is crucial for the organisms’ homeostasis. It is important to know the greatest number of factors affecting the protein regulation and determining the signal transduction pathway in the case of transmembrane proteins. Studying the role that water molecules play in these processes can lead to a better understanding of the basics of protein regulation at the molecular level.
This is especially important for Toll-like receptors. Any abnormality in the regulation of these proteins can cause serious diseases. Examination of the molecular aspects of the regulation of Toll-like receptors along with the characteristics of intermolecular interactions is, therefore, a key point that will help to understand their functioning.

Bzówka M’; Bagrowska W.; Góra A.; Recent Advances in Studying Toll-like Receptors with the Use of Computational Methods. J. Chem. Inf. Model.2023, 63, 12, 3669–3687 doi: 10.1021/acs.jcim.3c00419  ARTICLEOpen Access

Project is financed by:

grant no: 0141/DIA/2019/48

Rare diseases are mostly caused by small changes in DNA resulting in production of similar protein with disturbed functionality. A careful analysis of differences caused by a single mutation can provide priceless information for suffering people.

The genetic code is a set of rules used to translate the information encoded within the genetic material (DNA or RNA) into proteins. It is highly similar among all organisms. The code defines how codons specify particular amino acids which will be added consecutive during protein synthesis. A three-nucleotide codon in a nucleic acid sequence specifies a particular amino acid. Single nucleotide substitution (SNP) is a substitution of a single nucleotide at a specific position in the genome. SNPs might cause very subtle (i.e. silent or missense mutations) differences in the produced protein, while sometimes they result in a truncated, incomplete, and usually nonfunctional protein (i.e., nonsense mutation) by introducing a premature stop-codon into the transcribed mRNA.

Such substitutions might be the cause of several genetic disorders. Therefore, the RARE-ILLN project is run in close cooperation with the Maria Skłodowska-Curie National Research Institute of Oncology to ensure careful and detailed analysis. Using in silico methods, such as homology modeling, molecular dynamics simulations, and small molecules tracking approach, we are able to determine the structure of the protein of interests in which the substitution was find, and analyse the differences between the wild-type (protein without introduced substitution) and protein with introduced substitution that could be related with differences in proteins stability and functionality.

  1. Mitusińska K., et al. Structural analysis of the effect of Asn107Ser mutation on Alg13 activity and Alg13-Alg14 complex formation and expanding the phenotypic variability of ALG13-CDG. Biomolecules 2022, 12(3), 398 doi: 10.3390/biom12030398
  2. Jezela-Stanek A., et al. Proteins Structure Models in the Evaluation of Novel Variant (C.472_477del) in the MOSC2 Gene. Diagnostics 2020, 10, 821 doi:10.3390/diagnostics10100821

Water is called the “molecule of life”. What can we learn using it as a molecular probe during large macromolecules investigation?

The WAT-PROBE project aims to investigate the concept of protein structure characterisation by the intramolecular voids inspection approach. This conceptually straightforward idea can provide an alternative, easy method for protein structure-function relationship analysis. We are using water molecules as molecular probes for protein structure interior examination. With the use of a ligand tracking and local density analysis approach, we aim to confirm the usability of such an analysis for: hot-spot identification, cavity and tunnel description, and investigation of the dynamics of the protein interior. In parallel, we wish to improve the existing methods which can be applied to such analysis, and we are going to validate water models used in molecular dynamics simulations for protein interior investigation. The basic knowledge obtained in the project will be used to study the role of water molecules penetrating enzymes with regard to their selectivity and activity. The results of the in silico study will be validated with experimental techniques. 

The analysis of transport pathways and cavities with water as a molecular probe can provide useful information for protein engineering. The proper analysis of water ‘behaviour’ inside the protein core can significantly improve the description of intrinsic transport pathways with access to both geometrical and physico-chemical information including the energy profile, and the detection of hot-spots for protein redesign. The second applicability of the project is closely related to drug design and enzyme specificity optimisation. By mapping the dynamics of cavities, we wish to explore the potential of enzyme adaptation to different substrates/ligands. In this part of the project we are including calculations with organic co-solvents to get access to a description of non-bonding interactions. The validation will be validated in two aspects, the application of the method in protein reengineering, and pharmacophore description.

The expected outcomes of proposed project are:

  • further development of methods for small molecule tracking,
  • validation of the usability of water molecules as a molecular probe for the study of protein dynamics,
  • delivery of an alternative method with potential applicability in protein engineering and drug design,
  • description of the role of the water inside the protein core of enzymes. 

The results of hot-spot detection (A), exploration of cavities by water molecules (B), and identified water leakage (blue pathway) (C) obtained with a recent version of the AQUA-DUCT software [1] during Solanum tuberosum epoxide hydrolase study. Please note that besides gates (indicated by green and orange balls on picture A), other functionally important residues were also detected by AQUA-DUCT software, (Figures from [2]).
  1. Bzówka M. et al. Evolution of tunnels in α/β-hydrolase fold proteins—What can we learn from studying epoxide hydrolases? PLOS Computational Biology 2022, 18 (5), e1010119 doi: 10.1371/journal.pcbi.1010119  ARTICLEOpen Access
  2. Mitusińska K. et al. Geometry-Based versus Small-Molecule Tracking Method for Tunnel Identification: Benefits and Pitfalls. J. Chem. Inf. Model2022, 62, 24, 6803–6811. doi: 10.1021/acs.jcim.2c00985 ARTICLEOpen Access
  3. Mitusińka K. et al. Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network. Computational and Structural Biotechnology Journal 2022, 20, 193-205. doi: 10.1016/j.csbj.2021.10.042 ARTICLEOpen Access
  4. Mitusińska K., et al. AQUA-DUCT: Analysis of Molecular Dynamics Simulations of Macromolecules with the use of Molecular Probes [Article v1. 0]. Living Journal of Computational Molecular Science 2021, 2 (1), 21383 doi: 10.33011/livecoms.2.1.21383  ARTICLEOpen Access
  5. Magdziarz, T., et al. AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective. Bioinformatics 2020, 36(8), 2599-2601, doi:10.1093/bioinformatics/btz946 ARTICLEOpen Access
  6. Mitusińska K., et al. Applications of water molecules for analysis of macromolecule properties. Computational and Structural Biotechnology Journal 2020, 18, 355-365, doi:10.1016/j.csbj.2020.02.001 ARTICLEOpen Access
  7. Mitusińska, K., et al. Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking. Biomolecules 2018, 8, 143, doi:10.3390/biom8040143 ARTICLEOpen Access
  8. Magdziarz, T., et al. AQUA DUCT: A ligands tracking tool. Bioinformatics 2017, 33, 2045–2046, doi:10.1093/bioinformatics/btx125 ARTICLE

Software dedicated page:


Gatesunderestimated and quite often imperceptible “tools” of enzymes that appear to be one of the best candidates for modification of enzyme activity, selectivity and substrate specificity.

Research on enzymes conducted during the last decade provided evidences that regions located further from the active sites can also determine enzymes properties. Such control may come from co-reagents or inhibitors providing allosteric regulations or may appear as a result of localisation of active site deep into protein core. In second case transport occurs through tunnels network. Substrate access pathways provide additional constrains for binding of ligands to the active site. Precise control may be achieved by gates – dynamic systems made of individual amino acids residues, loops, secondary structure elements or domains, which are able to change a geometrical state between open and closed conformation reversibly and by such transition controls the flow of small molecules – substrates, products, ions and solvents – in and out of the protein structure.

The gates in enzymes functionally may:

  1. contribute to enzyme selectivity by controlling the access of substrates,
  2. protect a specific region of a protein against a solvent access or
  3. synchronize processes occurring in distinct parts of a protein.


The passage of molecules through the access pathways can be controlled by gates trough their specific molecular interactions, e.g., electrostatic, hydrophobic or their geometrical properties, e.g. size discrimination in the bottleneck. The proper function of the gates, even the simplest ones, may be indispensable for catalysis and the gating event can even represent the rate-limiting step of the catalytic cycle.

The expected outcomes of proposed project are:

  1. detailed description of the role of gating and anchoring residues in studied systems,
  2. identification of correlations between gating and anchoring residues,
  3. verification of hypothesis about higher rate of evolution of gating residues comparing with entire protein sequence,
  4. exploring the role of gating and anchoring residues in substrate/products transportation from/to active site of the enzyme,
  5. establishment of methodology for fast and accurate identification of gating and anchoring residues.


  1. Bzówka M., et al. Evolution of tunnels in α/β-hydrolase fold proteins—What can we learn from studying epoxide hydrolases? PLOS Computational Biology 2022, 18 (5), e1010119 doi: 10.1371/journal.pcbi.1010119 ARTICLEOpen Access
  2. Mitusińka K. et al. Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network. Computational and Structural Biotechnology Journal 2022, 20, 193-205. doi: 10.1016/j.csbj.2021.10.042 ARTICLEOpen Access
  3. Mitusińska K., et al. AQUA-DUCT: Analysis of Molecular Dynamics Simulations of Macromolecules with the use of Molecular Probes [Article v1. 0]. Living Journal of Computational Molecular Science 2021, 2 (1), 21383 doi: 10.33011/livecoms.2.1.21383
  4. Mitusińska, K., et al. Applications of water molecules for analysis of macromolecule properties. Computational and Structural Biotechnology Journal 18, 355–365 (2020).
  5. Magdziarz, T. et al. AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective. Bioinformatics (2019). doi:10.1093/bioinformatics/btz946
  6. Subramanian, K. et al. Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcus furiosus Phosphoglucose Isomerase. Biomolecules 9, 212 (2019)
  7. Mitusińska, K. et al. Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking. Biomolecules 8, 143 (2018)
  8. Płuciennik, A. et al. BALCONY: an R package for MSA and functional compartments of protein variability analysis. BMC Bioinformatics 19, 300 (2018)
  9. Subramanian, K. et al. Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access. PLoS One 13, e0198990 (2018)
  10. Magdziarz, T. et al. AQUA-DUCT: A ligands tracking tool. Bioinformatics 33, 2045–2046 (2017)

Project is financed by:


grant no. UMO-2013/10/E/NZ1/00649


We aim to build a prototype of the enzyme in which we can the activity switch on and off on request, or activate activity in a particular part of the living cell.

Project Partner – Loschmidt Laboratory, Brno, Czech Republic.

Enzymes are versatile proteins which catalyse most of reactions within the body. The structure of enzymes was optimised to perform catalysed reactions efficiently, with high activity and selectivity, during natural evolution. At the end of XIX century, the key-lock mechanism was proposed to explain the outstanding performance of enzymes. Specifically, close fitting of the substrate molecule (key) to the active centre of the enzyme (lock) was introduced to describe enzymatic properties. This simply and elegant theory works successfully in popular science until today, yet it fails when the active site is hidden deep within the protein core. Such enzymes, widely spread throughout the protein world, are equipped with tunnels possessing properties which can regulate the activity and selectivity of enzymes. These additional constraints provide an opportunity for control of reactivity and make enzymes with buried active sites ideal candidates for industrial and medical applications.

Most of the strategies proposed for enzyme redesign are focused on reengineering the vicinity of the active site. Such methods often result in loss of enzyme activity due to the rearrangement of residues that are crucial for the enzyme’s catalytic properties. Modification of the residues that build tunnels can provide a safe alternative to existing protein design protocols; however this requires a deep understanding of the transport phenomena through the tunnel network. Unfortunately due to the lack of experimental methods that can explore ligand transportation inside the protein core, the task of modifying tunnel residues is quite difficult to achieve.

To overcome challenges in this project we are employing a combination of modern computational tools which can visualize the protein exits and substrate entry points into the buried active site. State of the art methodology will be used to support experimentalists and to precisely describe ligand transportation phenomena. Due to the grouping of studied ligands into set of 30 different compounds we will identify subtle differences responsible for enzyme selectivity to aid our models.


Moreover to facilitate our research we have constructed an enzyme equipped with a switch located inside the tunnel. By changing reduction-oxidation conditions we are able to open or close the tunnel. This enzyme can be viewed as a prototype of a future enzyme in which the activity can be switched on/off on request or activated in a particular part of the living cell. During the project we will validate the possibility of our system grafting into other enzymes and the possibility of additional switch modifications to enhance control.

Our project is proceeding in cooperation with one of the best protein engineering groups – the Loschmidt Laboratory in Brno in Czech Republic – where most of the experimental work will be carried out.


  1. Raczyńska A. et al. Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity. PLoS ONE 2023 18(2), e0280776. doi: 10.1371/journal.pone.0280776 ARTICLEOpen Access
  2. Mitusińska, K., Skalski, T. & Góra, A. Simple Selection Procedure to Distinguish between Static and Flexible Loops. Int J Mol Sci 21, 2293 (2020)
  3. Mitusińska, K., Raczyńska, A., Bzówka, M., Bagrowska, W. & Góra, A. Applications of water molecules for analysis of macromolecule properties. Computational and Structural Biotechnology Journal 18, 355–365 (2020)
  4. Magdziarz, T. et al. AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective. Bioinformatics (2019). doi:10.1093/bioinformatics/btz946
  5. Brezovsky, J. et al. Engineering a de Novo Transport Tunnel. ACS Catal 6, 7597–7610 (2016)

Project is financed by:


grant no. UMO-2015/18/M/NZ1/00427


We aim to investigate molecular basis of the clinically observed correlations in patient response to cancer treatment by deep analysis of the influence of particular mutations into functionality of proteins involved in drug metabolism.

Single Nucleotide Polymorphisms (SNPs) are DNA sequence variations that can lead to the changes in the amino acid sequence of the coded protein (nonsynonymous SNPs). Such mutations can result in changes that are well tolerated in organism. However, they might play important role in specific circumstances, e.g. during intensive clinical therapy, affecting patient response to the treatment.


The current research on genetic polymorphisms apply statistical approaches to existing databases of various experimental data predominantly. Various high-throughput sequencing methods were applied to identify non-synonymous variants in the human genome and several databases were constructed to collect and examine the relationship between human genome sequence variation and the associated disease phenotypes. As a consequence a huge number of bioinformatics approaches were developed to predict functional and structural consequences of the SNPs. The quality of such an approach and the accuracy of the results corresponds to the quality of the collected observables and the database uniformity.

In our project we are exploring parallel path of genetic polymorphisms study – we aim to investigate molecular basis of the clinically observed correlations in patient response to cancer treatment by deep analysis of the influence of particular mutations into functionality of proteins involved in drug metabolism. Our aim is to propose modifications in treatment scheme leading to personalised medicine based on individual genetic pattern of the patient.

Past Projects

Past contributions of our team members.




Loschmidt Laboratories, Masaryk University, Brno, Czech Republic 2010 – 2013



Artur Góra


Modification of tunnels in haloalkane dehalogenazes (DatA, LinB) design of new mutants with modified activity and selectivity



Protein engineering can be applied for tailoring of naturally evolved enzymes. Modifications introduced to the residues forming the first or second shell of an active site currently represent the most common strategy for enzyme redesign. Recent project presents an example of engineering of substrate specificity of haloalkane dehalogenase by targeting the residues of their access tunnels.



Protein design was based on computational approach, with combination of QM/MM methods, MD and RAMD simulations, docking and software for tunnels detection (CAVER). Predicted mutants were constructed and their properties were analyzed by several experimental methods.



The haloalkane dehalogenases are the family of enzymes with buried active sites. Their activity and selectivity reflect the properties of the active site as well as substrate/product transport pathways. Project explores possibility of tunnels re-engineering in bacterial enzymes haloalkane dehalogenases. Based on the analysis carried out, new mutants of haloalkane dehalogenases LinB and DatA were designed with improved catalytic properties (activity and selectivity). Work provides evidence that careful redesign of access pathways represents a powerful strategy for the precise control of activity and selectivity of enzymes.



B. Kozlikova, E. Sebestova, V. Sustr, J. Brezovsky, O. Strnad, L. Daniel, D. Bednar, A. Pavelka, M. Manak, M. Bezdeka, P. Benes, M. Kotry, A. Gora, J. Damborsky, and J. Sochor, “CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures,” Bioinforma. Oxf. Engl., vol. 30, no. 18, pp. 2684–2685, Sep. 2014.

A. Gora, J. Brezovsky, and J. Damborsky, “Gates of Enzymes,” Chem. Rev., vol. 113, no. 8, pp. 5871–5923, 2013.

K. Hasan, A. Gora, J. Brezovsky, R. Chaloupkova, H. Moskalikova, A. Fortova, Z. Prokop, J. Damborsky – “Effect of Unique Halide-Stabilising Residue on the Catalytic Properties of Haloalkane Dehalogenase DatA from Agrobacterium tumefaciens C58” – FEBS J., vol. 280, no. 13, pp. 3149–3159, July 2013.

J. Brezovsky, E. Chovancova, A. Gora, A. Pavelka, L. Biedermannova, and J. Damborsky, “Software tools for identification, visualization and analysis of protein tunnels and channels,” Biotechnol. Adv., vol. 31, no. 1, pp. 38–49, Jan. 2013.ARTICLE

E. Chovancova, A. Pavelka, P. Benes, O. Strnad, J. Brezovsky, B. Kozlikova, A. Gora, V. Sustr, M. Klvana, P. Medek, L. Biedermannova, J. Sochor, and J. Damborsky, “CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures,” PLoS Comput Biol, vol. 8, no. 10, p. e1002708, Oct. 2012.ARTICLEOpen Access

L. Biedermannová, Z. Prokop, A. Gora, E. Chovancová, M. Kovács, J. Damborsky, and R. C. Wade, “A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB,” J. Biol. Chem., vol. 287, no. 34, pp. 29062–29074, Aug. 2012.ARTICLE






AIST Tohoku – Sendai, JAPAN,  2002/2003 and 2004-2006



Artur Góra



Dehydrogenation of metylocyclohexane into toluene



Storage of hydrogen in the form of chemical compounds can be an attractive alternative to traditional methods due to both economic and safety issue. One of the problems that still need to be solved is dehydrogenation of compounds and hydrogen recovery from storage compounds. The project aim development of methodology for easy hydrogen recovery and separation.



By the use of catalysts combined with modern membranes of high durability in low temperatures.



The use of compounds such as toluene (TOL), has been already shown to be potentially feasible for hydrogen storage for both mobile and stationary applications. The reversible reaction of dehydrogenation of MCH to toluene, and in general all dehydrogenation reactions of paraffins to aromatic compounds, is highly endothermic and strongly limited by thermodynamic equilibrium. The ability of the catalyst placed inside membrane tube combined with the selectivity of hydrogen permeation through the membrane makes it possible to remove hydrogen and thereby shift the reaction towards the product side. Careful study allow me to confirm high durability and stability of pore filled type of palladium membrane in reaction conditions in the low temperature range (100-300ºC). The new composition of membrane and location of palladium grains inside pores of ? alumina intermediate layer allowed to use palladium membranes in such drastic conditions without losing their selectivity. So far there was no any other reported information about membrane working in temperature below 300ºC for such a long time.



A. Gora, D. A. P. Tanaka, F. Mizukami, and T. M. Suzuki, “Lower temperature dehydrogenation of methylcyclohexane by membrane-assisted equilibrium shift,” Chem. Lett., vol. 35, pp. 1372–1373, 2006. ARTICLE





Institute of Catalysis and Surface Chemistry Polish Academy of Sciences and Jagiellonian University – Krakow, POLAND 2003-2005



Artur Góra



Study of colorizing and blanching phenomena in “smart windows”



Electrochromic devices can find application in “smart windows” fabrication, windows which are able to decrease the use of electricity consumption for air conditioning. The project aim investigations of the tungsten oxides role in the colorizing and blanching phenomena.



By theoretical methods (quantum chemical calculations – TD-DFT).



Electrochromic devices can find application in “smart windows” fabrication, windows which are able to decrease the use of electricity consumption for air conditioning. The purpose of the research was to investigate the mechanism of WO3 layer colorizing and blanching phenomena. To achieve this, the mechanism of Li ion transfer and its intercalation into WO3 bulk structure was studied by using advanced theoretical tools (TD-DFT methodology).



E. Broclawik, A. Gora, P. Liguzinski, P. Petelenz, and H. A. Witek, “Quantum chemical modeling of electrochromism of tungsten oxide films,” J. Chem. Phys., vol. 124, 2006. ARTICLE

E. Broclawik, A. Gora, P. Liguzinski, P. Petelenz, and M. Slawik, “Quantum chemical modelling of the process of lithium insertion intoWO(3) films,” Catal. Today, vol. 101, pp. 155–162, 2005. ARTICLE


SCR of Nitric Oxides



Jagiellonian University – Krakow, POLAND 1996-2002



Artur Góra



Study of vanadium-tungsten catalysts for DENOX process



Nitric oxides are important air pollutants. Vanadium-tungsten catalysts are commercially used for selective catalytic reduction (SCR) of NOx by ammonia, however the role of tungsten is unclear. The project aim investigations of the tungsten role in the increasing of the catalyst activity in low temperatures.



By the combination of experimental (TEM, Raman spectroscopy, vacuum equipment, GC/MS) and theoretical (quantum chemical calculations – DFT) methods.



Vanadium-tungsten catalysts are commercially used for selective catalytic reduction (SCR) of NOx by ammonia. The role of tungsten in decreasing of the onset temperature of the SCR of NOx remains unclear up to now. This role seems to be essential for better understanding of the catalyzed reaction and future development of more efficient catalysts. To clarify this, I have i) studied the interaction of water molecule with vanadia-tungsten catalyst surface and the creation of new active centers for SCR process by FT Raman spectroscopy and quantum chemical (DFT) modeling ii) successfully synthetized highly active vanadium-tungsten catalysts for SCR of NOx by NH3 by the use of sol-gel method. Extensive calculations for cluster models of active sites on the catalyst surface and their interaction with small substrate molecules showed possibility of the use of water molecules as a probe of acid-base properties of Lewis and Brønsted surface centers.



A. Gora and E. Broclawik, “Theoretical estimation of acid-base properties of Lewis and Bronsted centres at the V-W-O catalyst surface: water molecule as the probe in DFT calculations,” J. Mol. Catal. -Chem., vol. 215, pp. 187–193, 2004. ARTICLE

M. Najbar, A. Gora, A. Bialas, and A. Weselucha-Birczynska, “Low-temperature reactivity of the surface species of vanadia-tungsta catalyst,” Solid State Ion., vol. 141, pp. 499–506, 2001.ARTICLE

E. Broclawik, A. Gora, and M. Najbar, “The role of tungsten in formation of active sites for no SCR on the V-W-O catalyst surface – quantum chemical modeling (DFT),” J. Mol. Catal. -Chem., vol. 166, pp. 31–38, 2001. ARTICLE

M. Najbar, E. Broclawik, A. Gora, J. Camra, A. Bialas, and A. Weselucha-Birczynska, “Evolution of the surface species of the V2O5-WO3 catalysts,” Chem. Phys. Lett., vol. 325, pp. 330–339, 2000. ARTICLE

A. Gora, E. Broclawik, “Dissociation of the Water Molecule on the V-W-O Catalyst Surface – Quantum Chemical Modeling”, Polish Journal of Environmental Studies, 2000; 9(1):31-34.

A. Gora, E. Broclawik, and M. Najbar, “Quantum chemical modeling (DFT) of active species on the V-W-O catalyst surface in various redox conditions,” Comput. Chem., vol. 24, pp. 405–410, 2000. ARTICLE

M. Najbar, F. Mizukami, A. Bialas, J. Camra, A. Weselucha-Birczynska, H. Izutsu, and A. Gora, “Evolution of Ti-Sn-rutile-supported V2O5-WO3 catalyst during its use in nitric oxide reduction by ammonia,” Top. Catal., vol. 11, pp. 131–138, 2000. ARTICLE

M. Najbar, A. Bialas, F. Mizukami, A. Weselucha-Birczynska, E. Bielanska, A. Gora, “Vanadia-Tungsta DENOX Catalysts on High Surface Area Rutile”, Polish Journal of Environmental Studies, 1997; 6:83-88.


Last publication: Sci Rep 14, 13999 (2024). https://doi.org/10.1038/s41598-024-64806-x


Our Team Members Publications




PBP-A, a cyanobacterial DD-peptidase with high specificity for amidated muropeptides, exhibits pH-dependent promiscuous activity harmful to Escherichia coli

Gol Mohammad Dorrazehi, Matthias Winkle, Martin Desmet, Vincent Stroobant, Gamze Tanriver, Hervé Degand, Damien Evrard, Benoît Desguin, Pierre Morsomme, Jacob Biboy, Joe Gray, Karolina Mitusińska, Artur Góra, Waldemar Vollmer & Patrice Soumillion, ARTICLEOpen Access

Scientific Reports 14, 13999 (2024).



Penicillin binding proteins (PBPs) are involved in biosynthesis, remodeling and recycling of peptidoglycan (PG) in bacteria. PBP-A from Thermosynechococcus elongatus belongs to a cyanobacterial family of enzymes sharing close structural and phylogenetic proximity to class A β-lactamases. With the long-term aim of converting PBP-A into a β-lactamase by directed evolution, we simulated what may happen when an organism like Escherichia coli acquires such a new PBP and observed growth defect associated with the enzyme activity. To further explore the molecular origins of this harmful effect, we decided to characterize deeper the activity of PBP-A both in vitro and in vivo. We found that PBP-A is an enzyme endowed with DD-carboxypeptidase and DD-endopeptidase activities, featuring high specificity towards muropeptides amidated on the D-iso-glutamyl residue. We also show that a low promiscuous activity on non-amidated peptidoglycan deteriorates E. coli’s envelope, which is much higher under acidic conditions where substrate discrimination is mitigated. Besides expanding our knowledge of the biochemical activity of PBP-A, this work also highlights that promiscuity may depend on environmental conditions and how it may hinder rather than promote enzyme evolution in nature or in the laboratory.

Effects of γ-polyglutamic acid on grassland sandy soil properties and plant functional traits exposed to drought stress

Tomasz Skalski, Ewelina Zając, Elżbieta Jędrszczyk, Katarzyna Papaj, Joanna Kohyt, Artur Góra, Anna Kasprzycka, Divine Shytum, Barbara Skowera, Agnieszka Ziernicka-Wojtaszek, ARTICLEOpen Access

Scientific Reports 14, 3769 (2024).



The current study provides field experimental data that support the use of γ-polyglutamic acid (γ-PGA) in drought stress and proposes its application in grassland management. We hypothesized that water treatment combined with PGA application to sandy soil would reduce drought stress in grasslands more effectively than watering alone. A randomized block design was used, with three replicate watering blocks (no watering, weekly watering, and monthly watering) and PGA treatments at four different concentrations (0%, 0.3%, 1%, and 2% PGA). The results showed that PGA acts as a biostimulant, alleviating the effects of stress in plants by: (1) increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+/3+, Ca2+, and Mg2+, as well as N-NH4+, and N-NO3−, (2) elongating plant roots, (3) increasing the aboveground biomass, (4) improving the resprouting capacity of the dominant grass Nardus stricta, and (5) improving the regeneration of dicotyledons. In the case of meadows on sandy soils, the use of low PGA concentrations (0.3% or 1%) was the most beneficial for the availability of macro- and microelements and improving the functional traits of plants. Irrigation had a greater effect than using PGA only for the dicotyledon to monocotyledon ratio.




Recent Advances in Studying Toll-like Receptors with the Use of Computational Methods

Bzówka M, Bagrowska W, Góra A, ARTICLEOpen Access

J. Chem. Inf. Model., 2023, 63, 12, 3669–3687.



Toll-like receptors (TLRs) are transmembrane proteins that recognize various molecular patterns and activate signaling that triggers the immune response. In this review, our goal is to summarize how, in recent years, various computational solutions have contributed to a better understanding of TLRs, regarding both their function and mechanism of action. We update the recent information about small-molecule modulators and expanded the topic toward next-generation vaccine design, as well as studies of the dynamic nature of TLRs. Also, we underline problems that remain unsolved.

Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity

Raczyńska A, Kapica P, Papaj K, Stańczak A, Shyntum D, Spychalska P, Byczek-Wyrostek A, Góra A, ARTICLEOpen Access

PLoS ONE 18(2), e0280776.



The haloalkane dehalogenase LinB is a well-known enzyme that contains buried active site and is used for many modelling studies. Using classical molecular dynamics simulations of enzymes and substrates, we searched for transient binding sites on the surface of the LinB protein by calculating maps of enzyme-ligand interactions that were then transformed into sparse matrices. All residues considered as functionally important for enzyme performance (e.g., tunnel entrances) were excluded from the analysis to concentrate rather on non-obvious surface residues. From a set of 130 surface residues, twenty-six were proposed as a promising improvement of enzyme performance. Eventually, based on rational selection and filtering out the potentially unstable mutants, a small library of ten mutants was proposed to validate the possibility of fine-tuning the LinB protein. Nearly half of the predicted mutant structures showed improved activity towards the selected substrates, which demonstrates that the proposed approach could be applied to identify non-obvious yet beneficial mutations for enzyme performance especially when obvious locations have already been explored.




Evolution of tunnels in α/β-hydrolase fold proteins—What can we learn from studying epoxide hydrolases?

Bzówka M, Mitusińska K, Raczyńska A, Skalski T, Samol A, Bagrowska W, Magdziarz T, Góra A, ARTICLEOpen Access

PLOS Computational Biology 2022, 18 (5), e1010119.



The evolutionary variability of a protein’s residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to α/β-hydrolase fold proteins—one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels’ evolution analysis based on entropy values calculated for tunnels’ residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a ‘perforation’ mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzyme’s performance and can be applied widely for enzymes with buried active sites.

Geometry-Based versus Small-Molecule Tracking Method for Tunnel Identification: Benefits and Pitfalls.

Mitusińska K, Bzówka M, Magdziarz T, Góra A, ARTICLEOpen Access

J. Chem. Inf. Model. 2022, 62, 24, 6803–6811.



Different methods for tunnel identification, geometry-based and small-molecule tracking approaches, were compared to provide their benefits and pitfalls. Results obtained for both crystal structures and molecular dynamics (MD) simulations were analyzed to investigate if a more computationally demanding method would be beneficial. Careful examination of the results is essential for the low-diameter tunnel description, and assessment of the tunnel functionality based only on their geometrical parameters is challenging. We showed that the small-molecule tracking approach can provide a detailed description of the system; however, it can also be the most computationally demanding.

Structural Analysis of the Effect of Asn107Ser Mutation on Alg13 Activity and Alg13-Alg14 Complex Formation and Expanding the Phenotypic Variability of ALG13-CDG.

Mitusińska K, Góra A, Bogdańska A, Rożdżyńska-Świątkowska A, Tylki-Szymańska A, Jezela-Stanek A ARTICLEOpen Access

Biomolecules, 2022, 12(3) 398.



Congenital Disorders of Glycosylation (CDG) are multisystemic metabolic disorders showing highly heterogeneous clinical presentation, molecular etiology, and laboratory results. Here, we present different transferrin isoform patterns (obtained by isoelectric focusing) from three female patients harboring the ALG13 c.320A>G mutation. Contrary to other known variants of type I CDGs, where transferrin isoelectric focusing revealed notably increased asialo- and disialotransferrin fractions, a normal glycosylation pattern was observed in the probands. To verify this data and give novel insight into this variant, we modeled the human Alg13 protein and analyzed the dynamics of the apo structure and the complex with the UDP-GlcNAc substrate. We also modeled the Alg13-Alg14 heterodimer and ran multiple simulations of the complex in the presence of the substrate. Finally, we proposed a plausible complex formation mechanism.

Evaluation of Xa inhibitors as potential inhibitors of the SARS-CoV-2 Mpro protease.

Papaj K, Spychalska P, Kapica P, Fisher A, Nowak J, Bzówka M, Sellner M, Lill MA, Smieško M, Góra A ARTICLEOpen Access

PloS ONE 2022, 17(1):e0262482.



Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.

Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network.

Mitusińka K, Wojsa P, Bzówka M, Raczyńcsla A, Bagrowska W, Samol A, Kapica P, Góra A ARTICLEOpen Access

Computational and Structural Biotechnology Journal 20, 2022, 193-205.



Enzymes with buried active sites maintain their catalytic function via a single tunnel or tunnel network. In this study we analyzed the functionality of soluble epoxide hydrolases (sEHs) tunnel network, by comparing the overall enzyme structure with the tunnel’s shape and size. sEHs were divided into three groups based on their structure and the tunnel usage. The obtained results were compared with known substrate preferences of the studied enzymes, as well as reported in our other work evolutionary analyses data. The tunnel network architecture corresponded well with the evolutionary lineage of the source organism and large differences between enzymes were observed from long fragments insertions. This strategy can be used during protein re-engineering process for large changes introduction, whereas tunnel modification can be applied for fine-tuning of enzyme.



Investigation of Thiocarbamates as Potential Inhibitors of the SARS-CoV-2 Mpro.

Papaj K, Spychalska P, Hopko K, Kapica P, Fisher A, Lill MA, Bagrowska W, Nowak J, Szleper K, Smieško M, Kasprzycka A, Góra A ARTICLEOpen Access

Pharmaceuticals 2021, 14(11), 1153.



In the present study we tested, using the microscale thermophoresis technique, a small library of thionocarbamates, thiolocarbamates, sulfide and disulfide as potential lead compounds for SARS-CoV-2 Mpro drug design. The successfully identified binder is a representative of the thionocarbamates group with a high potential for future modifications aiming for higher affinity and solubility. The experimental analysis was extended by computational studies that show insufficient accuracy of the simplest and widely applied approaches and underline the necessity of applying more advanced methods to properly evaluate the affinity of potential SARS-CoV-2 Mpro binders.

Computational insights into the known inhibitors of human soluble epoxide hydrolase.

Bzówka M., Mitusińska K., Hopko K., Góra A. ARTICLEOpen Access

Drug Discovery Today, 26(8):1914-1921.




Human soluble epoxide hydrolase (hsEH) is involved in the hydrolysis of epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory properties. Given that EET conversion generates nonbioactive molecules, inhibition of this enzyme would be beneficial. Past decades of work on hsEH inhibitors resulted in numerous potential compounds, of which a hundred hsEH–ligand complexes were crystallized and deposited in the Protein Data Bank (PDB). We analyzed all deposited hsEH–ligand complexes to gain insight into the binding of inhibitors and to provide feedback on the future drug design processes. We also reviewed computationally driven strategies that were used to propose novel hsEH inhibitors.

AQUA-DUCT: Analysis of Molecular Dynamics Simulations of Macromolecules with the Use of Molecular Probes [Article v1.0].

Mitusińska K., Raczyńska A., Wojsa P., Bzówka M., Góra A. ARTICLEOpen Access

Living Journal of Computational Molecular Science 2 (1), 21383




AQUA-DUCT software reverses the standard approach of the molecular dynamics simulations analysis of macromolecules, focusing on solvent, cosolvent and small ligands analysis considered as specic molecular probes instead of analysis of macromolecules atoms movement. Here we present six basic tutorials instructing the users in the best practices for preparing, carrying out, and analysing AQUA-DUCT results in various applications. Users are expected to already have signicant experience with running standard molecular dynamics simulations in any dedicated software (e.g., Amber, GROMACS, NAMD), and usage of PyMOL visualising software. The tutorials range from a basic analysis of multiple solvents trajectory used for identication of the entries/exits to the protein core, to a complex one, like identication of the key hot-spots in cosolvent MD simulations that can be used as an insight for macromolecules description, analysis, re-engineering, and for drug design.

Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2.

Fisher A., Sellner M., Mitusińska K., Bzówka M., Lill M.A., Góra A., Smeisko M. ARTICLEOpen Access

International journal of molecular sciences 22 (4), 2065




The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (Mpro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARSCoV-2 Mpro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV Mpro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 Mpro inhibitors, as we report it, was not previously conducted.



Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is a Challenging Target for Small-Molecule Inhibitors Design.

Bzówka M., Mitusińska K., Raczyńska A., Samol A., Tuszyński J.A., Góra A. ARTICLEOpen Access

International journal of molecular sciences 21 (9), 3099




The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar SARS Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins show major differences in both shape and size indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site’s conformational changes during the simulation time indicates its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicates that the virus’ mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

Simple Selection Procedure to Distinguish between Static and Flexible Loops.

Mitusińska K., Skalski T., Góra A., Int. J. Mol. Sci. 2020, 21(7), 2293;ARTICLEOpen Access

International journal of molecular sciences 21 (7), 2293



Loops are the most variable and unorganized elements of the secondary structure of proteins. Their ability to shift their shape can play a role in the binding of small ligands, enzymatic catalysis, or protein–protein interactions. Due to the loop flexibility, the positions of their residues in solved structures show the largest B-factors, or in a worst-case scenario can be unknown. Based on the loops’ movements’ timeline, they can be divided into slow (static) and fast (flexible). Although most of the loops that are missing in experimental structures belong to the flexible loops group, the computational tools for loop reconstruction use a set of static loop conformations to predict the missing part of the structure and evaluate the model. We believe that these two loop types can adopt different conformations and that using scoring functions appropriate for static loops is not sufficient for flexible loops. We showed that common model evaluation methods, are insufficient in the case of flexible solvent-exposed loops. Instead, we recommend using the potential energy to evaluate such loop models. We provide a novel model selection method based on a set of geometrical parameters to distinguish between flexible and static loops without the use of molecular dynamics simulations. We have also pointed out the importance of water network and interactions with the solvent for the flexible loop modelling.

Applications of water molecules for analysis of macromolecule properties.

Mitusińska K., Raczyńska A., Bzówka M., Bagrowska W., Góra A.

Computational and Structural Biotechnology Journal 2020, 18, 355-365

https://doi:10.1016/j.csbj.2020.02.001 ARTICLEOpen Access


Water molecules maintain proteins’ structures, functions, stabilities and dynamics. They can occupy certain positions or pass quickly via a protein’s interior. Regardless of their behaviour, water molecules can be used for the analysis of proteins’ structural features and biochemical properties. Here, we present a list of several software programs that use the information provided by water molecules to: i) analyse protein structures and provide the optimal positions of water molecules for protein hydration, ii) identify high-occupancy water sites in order to analyse ligand binding modes, and iii) detect and describe tunnels and cavities. The analysis of water molecules’ distribution and trajectories sheds a light on proteins’ interactions with small molecules, on the dynamics of tunnels and cavities, on protein composition and also on the functionality, transportation network and location of functionally relevant residues. Finally, the correct placement of water molecules in protein crystal structures can significantly improve the reliability of molecular dynamics simulations.

Structure–bioavailability relationship study of genistein derivatives with antiproliferative activity on human cancer cell.

Papaj, K.; Kasprzycka, A.; Góra, A.; Grajoszek, A.; Rzepecka, G.; Stojko, J.; Barski, J.-J.; Szeja, W.; Rusin, A. J Pharm Biomed Anal 2020, 185, 113216;https:// doi:10.1016/J.JPBA.2020.113216 ARTICLEOpen Access


The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram′-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative’s biological properties.



AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective.

Magdziarz T., Mitusińska K., Bzówka M., Raczyńska A., Stańczak A., Banas M., Bagrowska W., Góra A. ,Bioinformatics 2019, 1–3. https://doi.org/10.1093/bioinformatics/btz946 ARTICLEOpen Access


Tunnels, pores, channels, pockets and cavities contribute to proteins architecture and performance. However, analysis and characteristics of transportation pathways and internal binding cavities are performed separately. We aimed to provide universal tool for analysis of proteins integral interior with access to detailed information on the ligands transportation phenomena and binding preferences.



Can a Mononuclear Iron(III)-Superoxo Active Site Catalyze the Decarboxylation of Dodecanoic Acid in UndA to Produce Biofuels?

Lin YT, Stańczak A, Manchev Y, Straganz GD, de Visser SP. Chemistry. 2019 Oct 4. https://doi.org/10.1002/chem.201903783 ARTICLEOpen Access


Decarboxylation of fatty acids is an important reaction in cell metabolism, but also has potential in biotechnology for the biosynthesis of hydrocarbons as biofuels. The recently discovered nonheme iron decarboxylase UndA is involved in the biosynthesis of 1-undecene from dodecanoic acid and using X-ray crystallography was assigned to be a mononuclear iron species. However, the work was contradicted by spectroscopic studies that suggested UndA to be more likely a dinuclear iron system. To resolve this controversy we decided to pursue a computational study on the reaction mechanism of fatty acid decarboxylation by UndA using iron(III)-superoxo and diiron(IV)-dioxo models. We tested several models with different protonation states of active site residues. Overall, however, the calculations imply that mononuclear iron(III)-superoxo is a sluggish oxidant of hydrogen atom abstraction reactions in UndA and will not be able to activate fatty acid residues by decarboxylation at room temperature. By contrast, a diiron-dioxo complex reacts with much lower hydrogen atom abstraction barriers and hence is a more likely oxidant in UndA.



Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcus furiosus Phosphoglucose Isomerase.

Subramanian, K.; Mitusińska, K.; Raedts, J.; Almourfi, F.; Joosten, H.-J.; Hendriks, S.; Sedelnikova, S.E.; Kengen, S.W.M.; Hagen, W.R.; Góra, A.; Martins dos Santos, V.A.P.; Baker, P.J.; van der Oost, J.; Schaap, P.J., Biomolecules 2019, 9, 212. https://doi.org/10.3390/biom9060212 ARTICLEOpen Access


The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.



Reaction mechanism between Cu(II)-enolate complex and O2 as a test case for methodology used in DFT computational studies.

A. Stańczak, A.Miłaczewska, T. Borowski, T. J Mol Model (2019) 25: 122. https://doi.org/10.1007/s00894-019-3998-3 ARTICLEOpen Access


The reaction mechanism of an intricate oxidation reaction of chlorodiketonate ligand of mononuclear Cu(II) complex was studied computationally employing five different models that differ in: a) basis set, b) the way that solvent corrections are included, and c) DFT functional. Qualitative and quantitative comparison of structures and enthalpy reaction profiles enabled us to assess how sensitive they are to the changes in computational methodology.




Exploring Solanum Tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking.

K. Mitusińska, T. Magdziarz, M. Bzówka, A. Stańczak, A. Góra, Biomolecules 2018, 8(4): 143. DOI: https://doi.org/10.3390/biom8040143 ARTICLEOpen Access


Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.



The modelling and enhancement of water hydrodynamics: general discussion.

Baaden, M.; Borthakur, M. P.; Casanova, S.; Coalson, R.; Freger, V.; Gonzalez, M.; Góra, A.; Hinds, B.; Hirunpinyopas, W.; Hummer, G.; Kumar, M.; Lynch, C.; Murail, S.; Noy, A.; Sansom, M.; Song, Q.; Vashisth, H.; Vögele, M., Faraday Discuss. 2018, 354–360, doi:10.1039/C8FD90021C. ARTICLE


Structure and function of natural proteins for water transport: General discussion.

Baaden, M.; Barboiu, M.; Bill, R. M.; Casanova, S.; Chen, C. L.; Conner, M.; Freger, V.; Gong, B.; Góra, A.; Hinds, B.; Horner, A.; Hummer, G.; Kumar, M.; Lokesh, M.; Mitra, S.; Noy, A.; Pohl, P.; Sadet, A.; Sansom, M.; Törnroth-Horsefield, S.; Vashisth, H., Faraday Discuss. 2018, 209, 83–95, doi:10.1039/C8FD90019A. ARTICLE



BALCONY: an R package for MSA and functional compartments of protein variability analysis.

A. Płuciennik, M. Stolarczyk, M. Bzówka, A. Raczyńska, T. Magdziarz, A. Góra, BMC Bioinformatics 2018, 19: 300. DOI:10.1186/s12859-018-2294-z. ARTICLEOpen Access


 Background: Here, we present an R package for entropy/variability analysis that facilitates prompt and convenient data extraction, manipulation and visualization of protein features from multiple sequence alignments. BALCONY can work with residues dispersed across a protein sequence and map them on the corresponding alignment of homologous protein sequences. Additionally, it provides several entropy and variability scores that indicate the conservation of each residue.

Results: Our package allows the user to visualize evolutionary variability by locating the positions most likely to vary and to assess mutation candidates in protein engineering.

Conclusion: In comparison to other R packages BALCONY allows conservation/variability analysis in context of protein structure with linkage of the appropriate metrics with physicochemical features of user choice.

Availability: CRAN project page: https://cran.r-project.org/package=BALCONY and our website: http://www.tunnelinggroup.pl/software/ for major platforms: Linux/Unix, Windows and Mac OS X.



Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access.

K. Subramanian, A. Góra, R. Spruijt, K. Mitusińska, M. Suarez-Diez, V. M. dos Santos, P. J. Schaap,  PLoS ONE 13(6): e0198990. DOI: 10.1371/journal.pone.0198990 ARTICLEOpen Access



D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.





AQUA-DUCT a ligands tracking tool.

T. Magdziarz, K. Mitusińska, S. Gołdowska, A. Płuciennik, M. Stolarczyk, M .Ługowska and A. Góra; Bioinformatics (2017) 33 (13): 2045-2046. DOI: 10.1093/bioinformatics/btx125 ARTICLE or PROOF VERSIONOpen Access


Motivation:The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA-DUCT.

Results: AQUA-DUCT is an easy-to-use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules e.g., water, oxygen, carbon dioxide, organic solvents, ions.

Platform and Availability: Linux, Windows, macOS, OpenBSD, http://www.aquaduct.pl

Contact: a.gora@tunnelinggroup.pl, info@aquaduct.pl

The available copy of paper is a pre-copyedited, author-produced version of an article accepted for publication in Bioinformatics following peer review. The version of record Bioinformatics (2017) 33 (13): 2045-2046. is available online at: https://academic.oup.com/bioinformatics/article-abstract/doi/10.1093/bioinformatics/btx125/3059930/AQUA-DUCT-a-ligands-tracking-tool?redirectedFrom=fulltext DOI: 10.1093/bioinformatics/btx125.



Engineering a de Novo Transport Tunnel ACS

J. Brezovsky, P. Babkova, O. Degtjarik, A. Fortova, A. Gora, I. Iermak, P. Rezacova, P. Dvorak, I. Smatanova, Z. Prokop, R. Chaloupkova, and J. Damborsky, Catalysis 2016, 6, 7597-7610 DOI: 10.1021/acscatal.6b02081 ARTICLE


Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. Absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional “hole” into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members, up to 32-times increased substrate inhibition and 17-times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to the native enzyme, and the most proficient haloalkane dehalogenase reported to date (kcat = 57 s-1 with 1,2-dibromoethane at 37oC and pH=8.6). Crystallographic analysis and molecular dynamics simulations confirmed successful introduction of structurally defined and functional transport tunnel. Our study demonstrates that whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels and we anticipate that this engineering strategy will facilitate creation of a wide range of useful biocatalysts.acs_catalysis_de_novo_tunnel


Molecular descriptor data explain market prices of a large commercial chemical compound library

J. Polanski, U. Kucia, R. Duszkiewicz, A. Kurczyk, T. Magdziarz and J. Gasteiger, Scientific Reports 6, Article number: 28521 (2016). doi:10.1038/srep28521 ARTICLEOpen Access


The relationship between the structure and a property of a chemical compound is an essential concept in chemistry srep28521-f1guiding, for example, drug design. Actually, however, we need economic considerations to fully understand the fate of drugs on the market. We are performing here for the first time the exploration of quantitative structure-economy relationships (QSER) for a large dataset of a commercial building block library of over 2.2 million chemicals. This investigation provided molecular statistics that shows that on average what we are paying for is the quantity of matter. On the other side, the influence of synthetic availability scores is also revealed. Finally, we are buying substances by looking at the molecular graphs or molecular formulas. Thus, those molecules that have a higher number of atoms look more attractive and are, on average, also more expensive. Our study shows how data binning could be used as an informative method when analyzing big data in chemistry.



CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures

B. Kozlikova, E. Sebestova, V. Sustr, J. Brezovsky, O. Strnad, L. Daniel, D. Bednar, A. Pavelka, M. Manak, M. Bezdeka, P. Benes, M. Kotry, A. Gora, J. Damborsky, and J. Sochor, Bioinforma. Oxf. Engl., vol. 30, no. 18, pp. 2684–2685, Sep. 2014 ARTICLE.


 The transport of ligands, ions or solvent molecules into proteins with buried binding sites or through the membrane is enabled by protein tunnels and channels. CAVER Analyst is a software tool for calculation, analysis and real-time visualization of access tunnels and channels in static and dynamic protein structures. It provides an intuitive graphic user interface for setting up the calculation and interactive exploration of identified tunnels/channels and their characteristics.


Gates of Enzymes

A. Gora, J. Brezovsky, and J. Damborsky, Chem. Rev., vol. 113, no. 8, pp. 5871–5923, 2013. ARTICLEOpen Access

Table of ContentsGATES_OF_ENZYMES

  1. Introduction
  2. Molecular Function of Gates
  3. Structural Basis of Gates
  4. Locations of Gates
  5. Engineering of Gates
  6. Conclusions


The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

K. Hasan, A. Gora, J. Brezovsky, R. Chaloupkova, H. Moskalikova, A. Fortova, Y. Nagata, J. Damborsky, and Z. Prokop, FEBS J., vol. 280, no. 13, pp. 3149–3159, July 2013.


FEBS2013 Haloalkane dehalogenases catalyse the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilising residues that are important in substrate binding and stabilisation of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenase, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 possesses a unique halide-stabilising tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the enzyme’s structure and catalytic properties were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen bonding patterns within the active sites of the wild-type and the mutant, and of the stabilisation of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding 3.7-fold, presumably due to the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the enzyme’s substrate specificity and reduced its K0.5 for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilises its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.


Software tools for identification, visualization and analysis of protein tunnels and channels

J. Brezovsky, E. Chovancova, A. Gora, A. Pavelka, L. Biedermannova, and J. Damborsky, Biotechnol. Adv., vol. 31, no. 1, pp. 38–49, Jan. 2013. ARTICLE


Protein structures contain highly complex systems of voids, making up specific features such as surface clefts or grooves, pockets, protrusions, cavities, pores or channels, and tunnels. Many of them are essential for the migration of solvents, ions and small molecules through proteins, and their binding to the functional sites. Analysis of these structural features is very important for understanding of structure-function relationships, for the design of potential inhibitors or proteins with improved functional properties. Here we critically review existing software tools specialized in rapid identification, visualization, analysis and design of protein tunnels and channels. The strengths and weaknesses of individual tools are reported together with examples of their applications for the analysis and engineering of various biological systems. This review can assist users with selecting a proper software tool for study of their biological problem as well as highlighting possible avenues for further development of existing tools. Development of novel descriptors representing not only geometry, but also electrostatics, hydrophobicity or dynamics, is needed for reliable identification of biologically relevant tunnels and channels.


A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB

L. Biedermannová, Z. Prokop, A. Gora, E. Chovancová, M. Kovács, J. Damborsky, and R. C. Wade, J. Biol. Chem., vol. 287, no. 34, pp. 29062–29074, Aug. 2012.ARTICLE



Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly JBC2012slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.


CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures

E. Chovancova, A. Pavelka, P. Benes, O. Strnad, J. Brezovsky, B. Kozlikova, A. Gora, V. Sustr, M. Klvana, P. Medek, L. Biedermannova, J. Sochor, and J. Damborsky, PLoS Comput Biol, vol. 8, no. 10, p. e1002708, Oct. 2012.ARTICLEOpen Access


 Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures.

caver_mHerein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.


Gora, A., Brezovsky, J. & Damborsky, J., Computer-assisted enzyme engineering by modification of tunnels, channels and gates. Current Opinion in Biotechnology vol 22, supplement 1 September 2011.

Gora, A., Pacheco Tanaka, D. A., Mizukami, F. & Suzuki, T. M., Low temperature hydrogen recovering from organic storage compounds – application of pore fill type palladium membrane. Proceedings of International Conference and Exhibition on Green Chemistry, 18-21 IX 2006 Kuala Lumpur, Malaysia, 2006.

Gora, A. & Brocławik E. Dissociation of the Water Molecule on the V-W-O Catalyst Surface – Quantum Chemical Modeling. Polish Journal of Environmental Studies, 9(1), 31-34, 2000.

Najbar M., Białas A., Mizukami F., Wesełucha-Birczyńska A., Bielańska E. & Gora A. Vanadia-Tungsta DENOX Catalysts on High Surface Area Rutile. Polish Journal of Environmental Studies, 6, 83-8, 1997.


Prokop Z, Gora A, Brezovsky J, Chalupkova R, Stepankova V & Damborsky J Protein Engineering Handbook, Volume 3: Book chapter: Engineering of protein tunnels: Keyhole-lock-key model for catalysis by the enzymes with buried active sites – ISBN 978-3-527-33123-9 – Wiley-VCH, Weinheim.


US Patent No 13/604,094 “Method of thermostabilization of a protein and/or stabilization towards organic solvents” Jiri Damborsky, Zbynek Prokop, Tana Koudelakova, Veronika Stepankova, Radka Chaloupkova, Eva Chovancova, Artur Wiktor Gora, Jan Brezovsky.


Information about software developed in our group.


AQUA-DUCT is a new tool facilitating analysis of the flow of solvent molecules in molecular dynamic simulations.

AQUA-DUCT allows extraction, analysis and visualization of the behaviour of solvent molecules during the entire simulation. Such analysis can be useful for the analysis of the enzymes with a buried active site, connected with surrounding solvent by tunnels. The water flow can be controlled by molecular properties of amino acids constituting tunnels or in more sophisticated enzymes by gates controlling the opening and closing of the access pathways. For more details please read our articles (AQβ, AQ1.0) or visit dedicated web page.

An example of AQUA-DUCT results. 
Approximated trajectories of all water molecules detected in active site cavity during MD simulations of M. musculus epoxide hydrolase.

How to cite:
1. T.Magdziarz , K.Mitusińska, M.Bzówka , A.Raczyńska ,A.Stańczak ,M.Banas, W.Bagrowska, A.Góra, AQUA-DUCT 1.0: structural and functional analysis of macromolecules from an intramolecular voids perspective.,  Bioinformatics 2019, 1–3. https://doi.org/10.1093/bioinformatics/btz946
2. T. Magdziarz et al., AQUA-DUCT a ligands tracking tool. Bioinformatics 2017 btx125. doi: 10.1093/bioinformatics/btx125

Better ALignment CONsensus analYsis

BALCONY is an R package that facilitates the evolutionary analysis and check the variability of selected amino acids in protein structure.

One of the unique functionalities of the BALCONY package concerns the analysis of individual amino acids in primary protein structure and their neighbours in tertiary structure. Residues defining active site cavities, co-factor binding sites, selectivity filters, channels or tunnels are examples of applications for this package.For installation please go to CRAN project page: BALCONY

How to cite: A. Płuciennik, M. Stolarczyk, M. Bzówka, A. Raczyńska, T. Magdziarz, A. Góra: BALCONY: an R package for MSA and functional compartments of protein variability analysis. BMC Bioinformatics 2018, 19: 300-307..



The recruitment procedure for new team members is constantly open. We are looking for highly motivated students interested in virtual screening and drug design, in chemical reaction modelling and in enzymology, reaction kinetics and protein inhibition study.

Both students from last years and just starting the study can apply. The best members can be awarded with individual stipends and will present their results on national and international conferences.

For more details please send your curriculum vitae and letter of application to:  Artur.Gora@polsl.pl.

Poszukujemy studentów zainteresowanych: wirtualnym screeningiem i projektowaniem leków, modelowaniem reakcji chemicznych; katalizą enzymatyczną oraz oddziaływaniami białko-ligand oraz rozwojem oprogramowania naukowego.

Mile widziane nie tylko osoby z ostatnich lat studiów, ale również zaczynające studia. Dla najlepszych osób przewidziane są stypendia studenckie; w ramach projektu będzie również możliwość przygotowania prac inżynierskich i magisterskich, prezentacja wyników na konferencjach zarówno krajowych jak i zagranicznych.

Zainteresowanych proszę o przesłanie CV oraz listu motywacyjnego na adres: Artur.Gora@polsl.pl


Tunneling Group
Biotechnology Centre
Silesian University of Technology
ul. Krzywoustego 8
44-100 Gliwice

Artur Góra, Ph.D.
phone: +48 322371659