A tetrapeptide fragment-based design method results in highly stable artificial proteins

Computational protein design has progressed rapidly over the last years. A number of design methods have been proposed and tested. In this paper, we report the successful application of a fragment-based method for protein design. The method uses statistical information on tetrapeptide backbone conformations. The previously published artificial fold of TOP 7 (Kuhlman et al., Science, 2003; 302:1364-1368) was chosen as template. A series of polypeptide sequences were created that were predicted to fold into this target structure. Two of the designed proteins, M5 and M7, were expressed and characterized by fluorescence spectroscopy, circular dichroism and NMR. They showed the hallmarks of well-ordered tertiary structure as well as cooperative folding/unfolding transitions. Furthermore, the two novel proteins were found to be highly stable against temperature and denaturant-induced unfolding.