During evolution of proteins from a common ancestor, one functional property can be preserved while others can vary leading to functional diversity. A systematic study of the corresponding adaptive mutations provides a key to one of the most challenging problems of modern structural biology - understanding the impact of amino acid substitutions on protein function. The subfamily-specific positions (SSPs) are conserved within functional subfamilies but are different between them and, therefore, seem to be responsible for functional diversity in protein superfamilies. Consequently, a corresponding method to perform the bioinformatic analysis of sequence and structural data has to be implemented in the common laboratory practice to study the structure-function relationship in proteins and develop novel protein engineering strategies. This paper describes Zebra web server - a powerful remote platform that implements a novel bioinformatic analysis algorithm to study diverse protein families. It is the first application that provides specificity determinants at different levels of functional classification, therefore addressing complex functional diversity of large superfamilies. Statistical analysis is implemented to automatically select a set of highly significant SSPs to be used as hotspots for directed evolution or rational design experiments and analyzed studying the structure-function relationship. Zebra results are provided in two ways - (1) as a single all-in-one parsable text file and (2) as PyMol sessions with structural representation of SSPs. Zebra web server is available at http://biokinet.belozersky.msu.ru/zebra .
Keywords: glutathione S-transferases; prediction of functional subfamilies; protein design and enzyme engineering; protein structure–function relationship; subfamily-specific positions.