Folding of a pressure-denatured model protein

Abstract

The noncovalent complex formed by the association of two fragments of chymotrypsin inhibitor-2 is reversibly denatured by pressure in the absence of chemical denaturants. On pressure release, the complex returned to its original conformation through a biphasic reaction, with firstorder rate constants of 0.012 and 0.002 s ؊1 , respectively. The slowest phase arises from an interconversion of the pressuredenatured state, as revealed by double pressure-jump experiments. Below 5 M, the process was concentration dependent with a second-order rate constant of 1,700 s ؊1 M ؊1 . Fragment association at atmospheric pressure showed a similar break in the order of the reaction above 5 M, but both first-and second-order folding͞association rates are 2.5 times faster than those for the refolding of the pressure-denatured state. Although the folding rates of the intact protein and the association of the fragments displayed nonlinear Eyring behavior for the temperature dependence, refolding of the pressure-denatured complex showed a linear response. The negligible heat capacity of activation ref lects a balance of minimal change in the burial of residues from the pressure-denatured state to the transition state. If we add the higher energy barrier in the refolding of the pressure-denatured state, the rate differences must lie in the structure of this state, which has to undergo a structural rearrangement. This clearly differs from the conformational f lexibility of the isolated fragments or the largely unfolded denatured state of the intact protein in acid and provides insight into denatured states of proteins under folding conditions. Kinetic studies aiming at the elucidation of protein-folding mechanisms are most frequently based on the reaction that takes place on the transfer of an unfolded polypeptide from strong denaturing conditions to those that favor a folded conformation. Pressure is an ideal perturbant for folding equilibria in that it gives valuable thermodynamic information without the need of altering the chemical composition of the solution. High-pressure techniques were applied in folding studies at the equilibrium (1-3) and more recently in the study of the transition state for protein folding (4). The possibility of monitoring folding kinetics by simply releasing the pressure is very attractive, and the method is particularly suitable for slow reactions. Pressure-jump relaxation kinetics was used to investigate the folding mechanism of pressure-denatured staphylococcal nuclease (5, 6). The effect of pressure on the rate of a reaction is described by (7) k p ϭ k 0 ⅐ewhere k p corresponds to the rate of the reaction at pressure p, k 0 , the rate at atmospheric pressure, and ⌬V ‡

The preaggregated state of an amyloidogenic protein: Hydrostatic pressure converts native transthyretin into the amyloidogenic state

“…tured tetramer. Denaturation without dissociation recently has been reported for other proteins (42,43).…”

Pressure-induced protein-folding/unfolding kinetics

“…To firmly establish the connection between this theoretical framework and reality, a generation of experiments have been devised to probe the details of the early folding events and to explore the topography of the folding landscape. A powerful technique that has received recent attention is the pressure dependence of protein-folding kinetics (1)(2)(3)(4)(5)(6). Developing the theoretical tools to interpret these pressure experiments on light of landscape theory is the focus of this paper.…”

Oscillatory Pressurization of an Animal Cell as a Poroelastic Spherical Body

“…78 High, static pressure is known to induce protein unfolding and to stabilize intermediate conformations of proteins. 43,49,61 The structure and properties of various biomembranes and the nucleoli also readapt under high static pressure. 46,63 It is unclear whether similar effects occur when high, cyclic pressure is applied on cells.…”