SUMMARY: Protein design requires information about how mutations affect protein stability. Many web-based predictors are available for this purpose, yet comparing them or using them en masse is difficult. Here, we present BenchStab, a console tool/Python package for easy and quick execution of 19 predictors and result collection on a list of mutants. Moreover, the tool is easily extensible with additional predictors. We created an independent dataset derived from the FireProtDB and evaluated 24 different prediction methods. AVAILABILITY AND IMPLEMENTATION: BenchStab is an open-source Python package available at https://github.com/loschmidt/BenchStab with a detailed README and example usage at https://loschmidt.chemi.muni.cz/benchstab. The BenchStab dataset is available on Zenodo: https://zenodo.org/records/10637728.

• MeSH
• databáze proteinů MeSH
• internet * MeSH
• proteiny chemie   MeSH
• software * MeSH
• stabilita proteinů MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny MeSH

Thermostable proteins find their use in numerous biomedical and biotechnological applications. However, the computational design of stable proteins often results in single-point mutations with a limited effect on protein stability. However, the construction of stable multiple-point mutants can prove difficult due to the possibility of antagonistic effects between individual mutations. FireProt protocol enables the automated computational design of highly stable multiple-point mutants. FireProt 2.0 builds on top of the previously published FireProt web, retaining the original functionality and expanding it with several new stabilization strategies. FireProt 2.0 integrates the AlphaFold database and the homology modeling for structure prediction, enabling calculations starting from a sequence. Multiple-point designs are constructed using the Bron-Kerbosch algorithm minimizing the antagonistic effect between the individual mutations. Users can newly limit the FireProt calculation to a set of user-defined mutations, run a saturation mutagenesis of the whole protein or select rigidifying mutations based on B-factors. Evolution-based back-to-consensus strategy is complemented by ancestral sequence reconstruction. FireProt 2.0 is significantly faster and a reworked graphical user interface broadens the tool's availability even to users with older hardware. FireProt 2.0 is freely available at http://loschmidt.chemi.muni.cz/fireprotweb.

• Klíčová slova
• B-factor, ancestral, back-to-consensus, epistasis, evolution, force-field, multiple-point mutant, protein engineering, saturation mutagenesis, thermostability,
• MeSH
• algoritmy * MeSH
• internet MeSH
• mutace MeSH
• proteiny * genetika   chemie   MeSH
• stabilita proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.

• Publikační typ
• časopisecké články MeSH

The majority of naturally occurring proteins have evolved to function under mild conditions inside the living organisms. One of the critical obstacles for the use of proteins in biotechnological applications is their insufficient stability at elevated temperatures or in the presence of salts. Since experimental screening for stabilizing mutations is typically laborious and expensive, in silico predictors are often used for narrowing down the mutational landscape. The recent advances in machine learning and artificial intelligence further facilitate the development of such computational tools. However, the accuracy of these predictors strongly depends on the quality and amount of data used for training and testing, which have often been reported as the current bottleneck of the approach. To address this problem, we present a novel database of experimental thermostability data for single-point mutants FireProtDB. The database combines the published datasets, data extracted manually from the recent literature, and the data collected in our laboratory. Its user interface is designed to facilitate both types of the expected use: (i) the interactive explorations of individual entries on the level of a protein or mutation and (ii) the construction of highly customized and machine learning-friendly datasets using advanced searching and filtering. The database is freely available at https://loschmidt.chemi.muni.cz/fireprotdb.

• MeSH
• anotace sekvence MeSH
• bodová mutace * MeSH
• databáze proteinů * MeSH
• datové soubory jako téma MeSH
• internet MeSH
• molekulární modely MeSH
• proteiny chemie   genetika   MeSH
• software MeSH
• stabilita proteinů MeSH
• strojové učení statistika a číselné údaje   MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH

There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot.

• MeSH
• Bacteria chemie   enzymologie   MeSH
• databáze proteinů MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• internet MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace * MeSH
• proteinové inženýrství metody   MeSH
• stabilita proteinů MeSH
• termodynamika MeSH
• uživatelské rozhraní počítače * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

• MeSH
• automatizace MeSH
• biokatalýza MeSH
• databáze proteinů MeSH
• internet * MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• mutace * MeSH
• mutageneze cílená metody   MeSH
• peptidová knihovna * MeSH
• proteiny chemie   genetika   MeSH
• software * MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peptidová knihovna * MeSH
• proteiny MeSH