Illustrative Tutorials for ProThermDB: Thermodynamic Database for Proteins and Mutants

ProThermDB (https://web.iitm.ac.in/bioinfo2/prothermdb/index.html) is a primary resource for protein stability, which contains experimentally determined thermodynamic data for proteins and their mutants. The most recent version of ProThermDB accumulates the data obtained from both high- and low-throughput experimental biophysical methods. It includes comprehensive information at four different levels, i.e.: (i) protein sequence and structure; (ii) experimental conditions; (iii) thermodynamic parameters such as Gibbs free energy, melting temperature, enthalpy, etc.; and (iv) literature. In the following protocols, we present detailed tutorials for retrieving data using different search, display and sorting options, interpretation of search results, description of each entry-level information category, data upload and download, cross-links with other databases, and visualization options. This protocol consists of six pictorial exercises, which are useful for biologists/users to understand the contents and organization of data in ProThermDB. Further, potential applications of ProThermDB in protein engineering are discussed. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Retrieval of experimental thermodynamic data for wild-type and mutants of a specific protein using a simple query. Basic Protocol 2: Retrieval of stabilizing point mutations, which are located at the interior of α-helical regions, and obtaining data by thermal denaturation methods. Basic Protocol 3: Retrieval of destabilizing point mutations, which are in β-sheets of exposed regions, and obtaining data by chemical denaturation methods (urea and GdnHCl). Basic Protocol 4: Retrieval of stabilizing and destabilizing point mutations in a range of physiological conditions (pH: 6-9 and T: 20°C-25°C) and publication years (2010-2020). Support Protocol: Downloading the entire data of the database for academic research purposes and submission of new data in ProThermDB.