Hierarchical versus non-hierarchical protein folding

Peter Flecker

Johannes Gutenberg Universität Mainz/FRG

The soybean Bowman Birk inhibitor (sBBI) is an attractive model protein for structural and mutational studies directed towards protein structure, folding and functionality. The parent protein of this study was a Lys16Arg and Met27Ile double replacement (termed as rBBI) of sBBI expressed in E. coli. A binary arrangement a trypsin- and a chymotrypsin-reactive subdomain in this protein was very useful for a detection of structural/functional irregularities transmitted from one subdomain into another by means of comparative titration and activity determination with trypsin and chymotrypsin. This can be used for a clear cut distinction between an independent (hierarchical) versus a coupled (non-hierarchical) folding of subdomains. In the present work we have studied the significance of a short b-turn forming segment near the exit of the trypsin binding loop of BBI. The mutants may be categorised into four classes according to the extent of the observed irregularities. The parent rBBI and one of the mutations distinguished themselves from the other variants by a spontaneous formation of two fully active subdomains upon refolding in solution. The other variants belonging to classes 2-4 were characterised by significant irregularities after refolding in solution. External assistance by means of Trypsin-Sepharose as a template with complementary structure was required with these mutants in order to obtain a fully active state of the variants. The class 2 mutations carrying hydrophobic residues of medium or large size gave only local irregularities in the trypsin-inhibitory region of the molecule and indistinguishable amounts and activities against chymotrypsin after refolding in solution and on the template. The amounts and the activity of the chymotrypsin-inhibitory subdomain were decreased or even completely abolished with the class 3 variants in accord with more global irregularities. The class 4 variants distinguished themselves from the class 2-3 variants by an unusual 2:1 stoichiometry with trypsin and chymotrypsin after template-directed folding. The mutants displaying local or more global irregularities could be distinguished by means of molecular modelling studies in accord with the experimental findings. The models seemed to be in line electrostatic interactions across the interdomain boundary and controlling a coupled folding of subdomains in accord with non-hierarchical models of protein folding.