Rosetta:MSF: a modular framework for multi-state computational protein design

Computational protein design (CPD) is a powerful technique to engineer existing proteins or to design novel ones that display desired properties. Rosetta is a software suite including algorithms for computational modeling and analysis of protein structures and offers many elaborate protocols created to solve highly specific tasks of protein engineering. Most of Rosetta’s protocols optimize sequences based on a single conformation (i. e. design state). However, challenging CPD objectives like multi-specificity design or the concurrent consideration of positive and negative design goals demand the simultaneous assessment of multiple states. This is why we have developed the multi-state framework MSF that facilitates the implementation of Rosetta’s single-state protocols in a multi-state environment and made available two frequently used protocols. Utilizing MSF, we demonstrated for one of these protocols that multi-state design yields a 15% higher performance than single-state design on a ligand-binding benchmark consisting of structural conformations. With this protocol, we designed de novo nine retro-aldolases on a conformational ensemble deduced from a (βα)8-barrel protein. All variants displayed measurable catalytic activity, testifying to a high success rate for this concept of multi-state enzyme design.Author summary: Protein engineering, i. e. the targeted modification or design of proteins has tremendous potential for medical and industrial applications. One generally applicable strategy for protein engineering is rational protein design: based on detailed knowledge of structure and function, computer programs like Rosetta propose the sequence of a protein possessing the desired properties. So far, most computer protocols have used rigid structures for design, which is a simplification because a protein’s structure is more accurately specified by a conformational ensemble. We have now implemented a framework for computational protein design that allows certain design protocols of Rosetta to make use of multiple design states like structural ensembles. An in silico assessment simulating ligand-binding design showed that this new approach generates more reliably native-like sequences than a single-state approach. As a proof-of-concept, we introduced de novo retro-aldolase activity into a scaffold protein and characterized nine variants experimentally, all of which were catalytically active.