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

Abstract

Modules, defined as stable, compact structure units in a globular protein, are good candidates for the construction of novel foldable proteins by permutation. Here we decomposed barnase into six modules (M1-M6) and constructed 23 barnase mutants containing permutations of the internal four (M2-M5) out of six modules. Globular proteins can also be subdivided into secondary structure units based on the extended structures that control the mutual relationships of the modules. We also decomposed barnase into six secondary structure units (S1-S6) and constructed 21 barnase mutants containing permutations of the internal four (S2-S5) out of six secondary structure units. Foldability of these two types of mutants was assessed by means of circular dichroism, fluorescence, and 1H-NMR measurements. A total of 15 of 23 module mutants and 15 of 21 secondary structure unit mutants formed definite secondary structures, such as α-helix and β-sheet, at 20 μM owing to intermolecular interactions, but most of them converted to random coil structures at a lower concentration (1 μM). Of the 44 mutants, only two, M3245 and S2543, gave distinct near-UV CD spectra. S2543 especially showed definite signal dispersion in the amide and methyl regions of the 1H-NMR spectrum, though M3245 did not. Furthermore, urea-induced unfolding of S2543 monitored by far-UV CD and fluorescence measurements showed a distinct cooperative transition. These results strongly suggest that S2543 takes partially folded conformations in aqueous solution. Our results also suggest that building 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 conformation, 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.