NMR-detected order in core residues of denatured bovine pancreatic trypsininhibitor

The NMR characteristics of [14-38](Abu), a synthetic variant of BPTI that i s partially folded in aqueous buffer near neutral pH, support a model of ea rly folding events which begin with stabilization of the nativelike, slow e xchange core [Barbar, E., Hare, M., Daragan, V., Barany, G., and Woodward, C. (1998) Biochemistry 37, 7822-7833 (1)]. In partially folded [14-38]Abu, urea denaturation profiles for representative amide protons show that globa l unfolding is non-two-state and that core residues require a higher concen tration of urea to unfold. Dynamic properties of pH-denatured [14-38](Abu) and fully reduced and unfolded BPTI analogue were determined from heteronuc lear NMR relaxation measurements at similar solution conditions. Difference s at various sites in the polypeptide chain were evaluated from spectral de nsity functions determined from T-1, T-2, and steady-state heteronuclear NO E data. Although denatured [14-38](Abu), contains no persistent secondary s tructure, its most ordered residues are those that, in native BPTI fold int o the slow exchange core. The fully reduced analogue is significantly more mobile and shows less heterogeneous dynamics, but at 1 degreesC, restricted motion is observed for residues in the central segments of the polypeptide chain. These observations indicate that there is a developing core or core s even in highly unfolded species. Apparently the effect of 14-38 disulfide on unfolded BPTI is to preferentially order and stabilize residues in the core.