Foldability of barnase mutants obtained by permutation of modules or secondary structure units

Modules, defined as stable, compact structure units in a globular protein, are good candidates for the construction of novel foldable proteins by perm utation. Here we decomposed barnase into six modules (M1-M6) and constructe d 23 barnase mutants containing permutations of the internal four (M2-M5) o ut of six modules. Globular proteins can also be subdivided into secondary structure units based on the extended structures that control the mutual re lationships of the modules. We also decomposed barnase into six secondary s tructure units (S1-S6) and constructed 21 barnase mutants containing permut ations of the internal four (S2-S5) out of six secondary structure units. F oldability of these two types of mutants was assessed by means of circular dichroism, fluorescence, and H-1-NMR measurements. A total of 15 of 23 modu le mutants and 15 of 21 secondary structure unit mutants formed definite se condary structures, such as alpha-helix and beta-sheet, at 20 mu M owing to intermolecular interactions, but most of them converted to random coil str uctures at a lower concentration (1 mu M). Of the 44 mutants, only two, M32 45 and S2543, gave distinct near-UV CD spectra. S2543 especially showed def inite signal dispersion in the amide and methyl regions of the H-1-NMR spec trum, though M3245 did not. Furthermore, urea-induced unfolding of S2543 mo nitored by far-UV CD and fluorescence measurements showed a distinct cooper ative transition. These results strongly suggest that S2543 takes partially folded conformations in aqueous solution. Our results also suggest that bu ilding blocks such as secondary structure units capable of taking different stable conformations by adapting themselves to the surrounding environment , rather than building blocks such as modules having a specified stable con formation, are required for the formation of foldable proteins. Therefore, the use of secondary structure units for the construction of novel globular proteins is likely to be an effective approach. (C) 1999 Academic Press.