Partially folded intermediates during trypsinogen denaturation

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure

In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures). Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biologicalphenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.