Partially folded intermediates during trypsinogen denaturation.

N.F Martins,M.M Santoro

doi:10.1590/s0100-879x1999000600002

Abstract

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 delta GH2O = 6.99 +/- 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 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 A to 26.0 +/- 0.3 A 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 A to 25.7 +/- 0.6 A 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.

Highlights

Protein folding was generally believed to be a highly cooperative two-state process in which only the native and completely unfolded states were significantly populated with no apparent intermediates
The unfolding of trypsinogen induced by guanidine chloride, monitored in the tertiary structure, showed quite coincident data, suggesting a highly cooperative denaturation fitting a two-state model (Figure 1)
These results suggest that the denaturation of trypsinogen consists of two successive phases - the first with loss of tertiary structure and exposure of hydrophobic residues reaching an intermediate state, followed by a second phase with loss of secondary structure reaching the completely unfolded state

Summary

Introduction

Protein folding was generally believed to be a highly cooperative two-state process in which only the native and completely unfolded states were significantly populated with no apparent intermediates. Of special interest in understanding the factors that determine the three-dimensional structure of globular proteins is the detection and identification of intermediates in folding reactions, since recent equilibrium and kinetic studies have shown the existence of stable intermediate conformational states for several proteins [1,2]. Later kinetic experiments demonstrated for several other proteins that denaturation can occur through another compact intermediate, named pre-molten globule [7,8] This state has a substantial amount of fluctuating secondary structure, is partly compact and has solvent-accessible non-polar clusters. This evidence suggests that the molten globule and other intermediates are obligatory in the folding pathway, their exact significance in the mechanism of folding is still not understood [9]

Methods

Results

Conclusion