Peter Flecker, Johannes Gutenberg University, Department of Chemistry and Pharmacy, Duesbergweg 10-14, D-55128 Mainz, FRG. E-mail:


All information for the three dimensional structure of proteins and their functionality is encrypted within their amino acid sequences. The natural amino acid sequences of proteins have been perfected by evolution not only for their functional structure but also for a rapid and highly directional acquisition of their folded, fully functional state. An unambiguous distinction between these two possibilities is important for a clear-cut interpretation of consequences of amino acid replacements in protein engineering experiments.

The double-headed Bowman Birk serine protease inhibitor (BBI) built up from two triple stranded b-hairpin domains directed against trypsin and chymotrypsin was selected as a model protein. The double-headed arrangement of two independent subdomains facilitates the detection of long-range irregularities transmitted from the trypsin- into the chymotrypsin-inhibitory region used as a reference.



The exposed hydrophobic patch belonging to the trypsin-inhibitory region (Tr) is shown in ochre and that projecting out from the chymotrypsin-inhibitory region (Ch) is shown in yellow. Residues belonging to the buried polar interior are highlighted in CPK.   


The exposed hydrophobic patches on the protein surface and a polar protein interior appear as structural peculiarities more reminiscent of the kind of structural features that have been postulated to occur in partially folded proteins rather than their folded state.

The autonomous refolding competence of the parent protein was abolished as s result of amino acid replacements, resulting in heterogeneous populations of conformers greatly differing in their activity. In contrast to the autonomous refolding competence of the parent protein, the variants require the presence of trypsin-Sepharose as a template with complementary structure in order to reach their fully active state. The fully active state of the variants attained by means of this method returns to the initial mixture of conformers upon subsequent incubation in the refolding buffer in a slow first order reaction. Therefore, the fully active state of the variants may be regarded as local energetic minima surrounded by high barriers of activation. The appearance of apparently metastable state supports a kinetic reaction control for the variants on the template although it cannot rule out a thermodynamic reaction control. In fact, the template facilitates folding not only kinetically, by reducing the high barrier of activation in solution but also thermodynamically by stabilising the fully active state by means of protein-protein-interactions. Protein metastability has also been documented for certain proteolytic enzymes after removal of their prosequences and for the native conformation of viral hemagglutinins. Presently, it is unknown whether the native conformation of soybean BBI corresponds to a global energy minimum or a metastable state on its conformational landscape. However, the inside-out situation in BBI and the conformational changes that are induced with reducing agents even in the absence of denaturants seem to be more in favour of the second possibility.