Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps

Abstract

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (DeltaH(#)), and activation entropy (DeltaS(#)) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative DeltaH(#) of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro(114) isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the beta-hairpin comprising Trp(115). A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.

FIGURE 1

Schematic representation of the mT-jump apparatus. The instrument, installed on a Bio-Logic stopped-flow basis, is fully controlled by the manufacturer's software and achieves temperature changes by mixing two solutions of initially different temperatures, contained in two syringes (1). Three independent thermoelectric elements (2) are used to control the temperatures of the two storage lines (4) and of the observation cell (3). The storage lines are built into aluminum blocks in direct contact with the Peltier elements. Their volume is sufficient for one stopped-flow experiment. The temperature of the observation cell is monitored by a temperature probe (5) attached to its quartz surface.

FIGURE 2

Fluorescence emission spectra and thermal transition curves of RNase A. (A) Wild-type protein. (B) Y115W variant. Spectra under native and unfolded conditions are represented as solid and dashed lines, respectively. Protein concentration was 0.1 mg ml⁻¹ in sodium acetate buffer, 50 mM, pH 5.0. (Insets) Temperature-induced unfolding curves. Solid lines are nonlinear least-squares fits based on a two-state model (Eq. 1).

FIGURE 3

T-jump induced unfolding/folding relaxation kinetics of RNase A Y115W. (A) Heating T-jumps started from 45°C. They reached 49, 51, 53, 55, 57, 59, 61, and 63°C. (B) Cooling T-jumps started from 63°C. They reached 59, 57, 55, 53, 51, 49, 47, and 45°C. Solution conditions: Y115W at 0.05 mg ml⁻¹ in sodium acetate buffer, 50 mM, pH 5.0.

FIGURE 4

Temperature dependence of the measured rate constant (k_obs). k_obs was determined from heating (triangles) and cooling (inverted triangles) T-jumps, for wild-type RNase A (A) and its Y115W variant (B). For the latter, a fast (open triangles) and a slow (solid triangles) kinetic phase were observed. k_obs obtained from heating and cooling T-jumps of different magnitude (8, 15, and 22°C), but reaching the same final temperature, are represented as horizontal traits inside circles.

FIGURE 5

Temperature dependence of the individual rate constants (k_f, k_u). (A) k_f (open symbols) and k_u (solid symbols) determined from heating (triangles) and cooling (inverted triangles) T-jumps. The protein was wild-type RNase A. (B) k_f (open symbols) and k_u (solid symbols) for the fast (circles) and slow (squares) kinetic phases determined from cooling T-jumps. The protein was the RNase A variant Y115W.

FIGURE 6

Path-dependent Arrhenius plots. k_f (open symbols) and k_u (solid symbols) were determined from heating (circles) and cooling (squares) T-jumps. The protein was the RNase A variant Y115W. Only the slow kinetic phase is shown.

FIGURE 7

Kinetic transition states of the temperature-induced folding/unfolding reaction of RNase A. Changes of entropy (A) and enthalpy (B) were determined from the temperature dependence of k_f and k_u of both heating and cooling T-jumps of wild-type RNase A (left) and its Y115W variant (right). The direction of the T-jumps is indicated by arrows. Thermodynamic parameters of the folded state were set to zero. U and N denote the unfolded and native states, respectively. # represents the TS ensemble.

FIGURE 8

Kinetic reaction model for the temperature-triggered folding/unfolding of RNase A. The shaded area indicates the fast kinetic phases observed only for the Y115W variant. Solid and shaded arrows denote heating and cooling T-jumps, respectively. Undetectable kinetic phases corresponding to conformational unfolding/folding are denoted by the black vertical shape at left.