Aleksandr Popov, Svetlana Dubiley and Konstantin Severinov co-authored a preprint “Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2” that has been recently released at bioRxiv. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The researchers performed a rabid semi-comprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (TfuB2) using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2 software. Analysis of 34 TfuB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, TfuB1. Full text of the preprint is available here.