UNFOLDING AND REFOLDING OF THE NATIVE STRUCTURE OF BOVINE PANCREATIC TRYPSIN-INHIBITOR STUDIED BY COMPUTER-SIMULATIONS

A new procedure for studying the folding and unfolding of proteins, wi th an application to bovine pancreatic trypsin inhibitor (BPTI), is re ported. The unfolding and refolding of the native structure of the pro tein are characterized by the dimensions of the protein, expressed in terms of the three principal radii of the structure considered as an e llipsoid. A dynamic equation, describing the variations of the princip al radii on the unfolding path, and a numerical procedure to solve thi s equation are proposed. Expanded and distorted conformations are refo lded to the native structure by a dimensional-constraint energy minimi zation procedure. A unique and reproducible unfolding pathway for an i ntermediate of BPTI lacking the [30,51] disulfide bond is obtained. Th e resulting unfolded conformations are extended; they contain near-nat ive local structure, but their longest principal radii are more than 2 .5 times greater than that of the native structure. The most interesti ng finding is that the majority of expanded conformations, generated u nder various conditions, can be refolded closely to the native structu re, as measured by the correct overall chain fold, by the rms deviatio ns from the native structure of only 1.9-3.1 angstrom, and by the ener gy differences of about 10 kcal/mol from the native structure. Introdu ction of the [30,51] disulfide bond at this stage, followed by minimiz ation, improves the closeness of the refolded structures to the native structure, reducing the rms deviations to 0.9-2.0 angstrom. The uniqu e refolding of these expanded structures over such a large conformatio nal space implies that the folding is strongly dictated by the interac tions in the amino acid sequence of BPTI. The simulations indicate tha t, under conditions that favor a compact structure as mimicked by the volume constraints in our algorithm; the expanded conformations have a strong tendency to move toward the native structure; therefore, they probably would be favorable folding intermediates. The results present ed here support a general model for protein folding, i.e., progressive formation of partially folded structural units, followed by collapse to the compact native structure. The general applicability of the proc edure is also discussed.