The quest to predict and understand protein evolution has been hindered by limitations on both the theoretical and the experimental fronts. Most existing theoretical models of evolution are descriptive, rather than predictive, leaving the final modifications in the hands of researchers. Existing experimental techniques to help probe the evolutionary sequence space of proteins, such as directed evolution, are resource-intensive and require specialised skills. We present the successor sequence predictor (SSP) as an innovative solution. Successor sequence predictor is an in silico protein design method that mimics laboratory-based protein evolution by reconstructing a protein's evolutionary history and suggesting future amino acid substitutions based on trends observed in that history through carefully selected physicochemical descriptors. This approach enhances specialised proteins by predicting mutations that improve desired properties, such as thermostability, activity, and solubility. Successor Sequence Predictor can thus be used as a general protein engineering tool to develop practically useful proteins. The code of the Successor Sequence Predictor is provided at https://github.com/loschmidt/successor-sequence-predictor , and the design of mutations will be also possible via an easy-to-use web server https://loschmidt.chemi.muni.cz/fireprotasr/ . SCIENTIFIC CONTRIBUTION: The Successor Sequence Predictor advances protein evolution prediction at the amino acid level by integrating ancestral sequence reconstruction with a novel in silico approach that models evolutionary trends through selected physicochemical descriptors. Unlike prior work, SSP can forecast future amino acid substitutions that enhance protein properties such as thermostability, activity, and solubility. This method reduces reliance on resource-intensive directed evolution techniques while providing a generalizable, predictive tool for protein engineering.

• Klíčová slova
• Activity, Adaptation, Evolution, Evolutionary trajectory, Protein design, Solubility, Thermostability,
• Publikační typ
• časopisecké články MeSH

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

Recent progress in engineering highly promising biocatalysts has increasingly involved machine learning methods. These methods leverage existing experimental and simulation data to aid in the discovery and annotation of promising enzymes, as well as in suggesting beneficial mutations for improving known targets. The field of machine learning for protein engineering is gathering steam, driven by recent success stories and notable progress in other areas. It already encompasses ambitious tasks such as understanding and predicting protein structure and function, catalytic efficiency, enantioselectivity, protein dynamics, stability, solubility, aggregation, and more. Nonetheless, the field is still evolving, with many challenges to overcome and questions to address. In this Perspective, we provide an overview of ongoing trends in this domain, highlight recent case studies, and examine the current limitations of machine learning-based methods. We emphasize the crucial importance of thorough experimental validation of emerging models before their use for rational protein design. We present our opinions on the fundamental problems and outline the potential directions for future research.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Protein solubility is an attractive engineering target primarily due to its relation to yields in protein production and manufacturing. Moreover, better knowledge of the mutational effects on protein solubility could connect several serious human diseases with protein aggregation. However, we have limited understanding of the protein structural determinants of solubility, and the available data have mostly been scattered in the literature. Here, we present SoluProtMutDB - the first database containing data on protein solubility changes upon mutations. Our database accommodates 33 000 measurements of 17 000 protein variants in 103 different proteins. The database can serve as an essential source of information for the researchers designing improved protein variants or those developing machine learning tools to predict the effects of mutations on solubility. The database comprises all the previously published solubility datasets and thousands of new data points from recent publications, including deep mutational scanning experiments. Moreover, it features many available experimental conditions known to affect protein solubility. The datasets have been manually curated with substantial corrections, improving suitability for machine learning applications. The database is available at loschmidt.chemi.muni.cz/soluprotmutdb.

• Klíčová slova
• Machine learning, Mutational database, Protein aggregation, Protein engineering, Protein yield, Soluble expression,
• Publikační typ
• časopisecké články MeSH

The importance of the quantitative description of protein unfolding and aggregation for the rational design of stability or understanding the molecular basis of protein misfolding diseases is well established. Protein thermostability is typically assessed by calorimetric or spectroscopic techniques that monitor different complementary signals during unfolding. The CalFitter webserver has already proved integral to deriving invaluable energy parameters by global data analysis. Here, we introduce CalFitter 2.0, which newly incorporates singular value decomposition (SVD) of multi-wavelength spectral datasets into the global fitting pipeline. Processed time- or temperature-evolved SVD components can now be fitted together with other experimental data types. Moreover, deconvoluted basis spectra provide spectral fingerprints of relevant macrostates populated during unfolding, which greatly enriches the information gains of the CalFitter output. The SVD analysis is fully automated in a highly interactive module, providing access to the results to users without any prior knowledge of the underlying mathematics. Additionally, a novel data uploading wizard has been implemented to facilitate rapid and easy uploading of multiple datasets. Together, the newly introduced changes significantly improve the user experience, making this software a unique, robust, and interactive platform for the analysis of protein thermal denaturation data. The webserver is freely accessible at https://loschmidt.chemi.muni.cz/calfitter.

• MeSH
• denaturace proteinů MeSH
• proteiny * chemie   MeSH
• rozbalení proteinů * MeSH
• software MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH