A novel protocol for the design of artificial (β/α)8-barrel proteins

The design of protein de novo is an emerging field in biochemistry, where artificial proteins are first designed in silico and then validated experimentally. This research, which rests mainly on our current understanding of protein structure, function, folding, stability and solubility, contributes to expand our knowledge of proteins in general.
Our group has a long tradition in the design of artificial (β/α)8 -barrel proteins (called Octarellins). This fold is extremely interesting because it is widespread in nature (10% of the known proteins contain this fold) and in catalysis (it is present in 5/6 classes of enzyme). Here we present a protocol to design de novo (β/α)8-barrels with the more recent and best performing tools: Rosetta and Modeller (modelling softwares), and GROMACS (molecular dynamic simulations).
First, 4000 artificial backbone structures were created with the use of modelling packages Rosetta and Modeller. 54 out of them were selected as targets for the following steps of sequence design and energy minimization (10 cycles), in order to find the best sequence to fit each target. More than 10000 different artificial sequences were created. Selection steps were performed in order to reduce the number of candidates for each target and the best ones were subjected to molecular dynamic simulation.
Among this, 5 models were finally chosen for gene synthesis and experimental validation, and are currently being tested for expression in E. coli and preliminary purification.