Characterization of Stability and Foldedness in the NextGen Right-Handed Four-Helix Bundle Library

Abstract

The de novo design of proteins, using novel amino acid sequences, has been proposed as a means of expanding the limited enzymatic activities and corresponding protein structures proposed by nature. To that end, design of well-folded, stable protein structures, considered key to efficient and selective activity, is necessary. Here it is reported the characterization of a new library of right-handed four-helix bundles (R4HB) designed de novo using a combinatorial approach. A screen for foldedness using C-terminal fusion of folding reporter green fluorescent protein (FRGFP) identified several stable and well-folded proteins as determined by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. Eight proteins characterized by CD formed helical bundles and had denaturation midpoints (T\(_{M}\)) above 76\(^{o}\) C. One construct, R4HB01, was founded to have a (T\(_{M}\)) of 145\(^{o}\)C and a maximum free energy of 9.878 kcal/mol; another, R4HB10, gave well-resolved peaks by NMR, suggesting good stability and rigidity. Though only a handful of proteins were characterized, the consistent finding of impressive stability properties motivates deeper analysis of the library, including whether improved stability imparts improved activity in functional assays.