Probing the folding intermediate of Rd-apocyt b562 by protein engineering and infrared T-jump

Abstract

Small proteins often fold in an apparent two-state manner with the absence of detectable early-folding intermediates. Recently, using native-state hydrogen exchange, intermediates that exist after the rate-limiting transition state have been identified for several proteins. However, little is known about the folding kinetics from these post-transition intermediates to their corresponding native states. Herein, we have used protein engineering and a laser-induced temperature-jump (T-jump) technique to investigate this issue and have applied it to Rd-apocyt b(562) , a four-helix bundle protein. Previously, it has been shown that Rd-apocyt b(562) folds via an on-pathway hidden intermediate, which has only the N-terminal helix unfolded. In the present study, a double mutation (V16G/I17A) in the N-terminal helix of Rd-apocyt b(562) was made to further increase the relative population of this intermediate state at high temperature by selectively destabilizing the native state. In the circular dichroism thermal melting experiment, this mutant showed apparent two-state folding behavior. However, in the T-jump experiment, two kinetic phases were observed. Therefore, these results are in agreement with the idea that a folding intermediate is populated on the folding pathway of Rd-apocyt b(562) . Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 microsec)(-1) at 65 degrees C.

Figure 1.

A qualitative illustration of the protein-engineering approach used in the present study, wherein destabilizing mutations were introduced to enhance the population of the intermediate state at high temperature. The curves represent the temperature-dependent free energies of the unfolded and intermediate states, relative to that of the folded state. (A) Wild-type Rd-apocyt b₅₆₂, (B) Mutant-1, (C) Mutant-2 (i.e., the mimic of the folding intermediate). In C, the dashed line represents the hypothetical native state, which is significantly destabilized and not populated under the conditions of these experiments. (D) Cartoon representations of the structures of the native (N) and intermediate (I) states of Rd-apocyt b₅₆₂, where the red helix is at the N terminus.

Figure 2.

Temperature-dependent mean residue ellipticities at 222 nm of Rd-apocyt b₅₆₂ (+), Mutant-1 (▵), and Mutant-2 (○). Solid lines are fits to the corresponding experimental data according to the method described in the text.

Figure 3.

(Bottom panel) A representative T-jump-induced relaxation trace of Rd-apocyt b₅₆₂ measured at 1632 cm⁻¹. The T-jump was from 72.1°C to 83.9°C. The smooth line is the fit to the following single-exponential function: ΔOD(t) = A × [1 − B × exp(−t/τ)], with A = −0.025, B = 0.53, and τ = 79.3 μsec. (B) Shown are the residuals of the fit. (A) Also shows the residuals of the fit to the following double-exponential function: ΔOD(t) = A × [1 − B₁ × exp(−t/τ₁) − B₂ × exp(−t/τ₂)], with A = −0.025, B₁ = 0.48, B₂ = 0.08, τ₁ = 86.1 μsec, and τ₂ = 17.3 μsec.

Figure 4.

(Bottom panel) A representative T-jump-induced relaxation trace of Mutant-1 measured at 1632 cm⁻¹. The T-jump was from 55.7°C to 64.8°C. The smooth line is the fit to the following double-exponential function: ΔOD(t) = A × [1 − B₁ × exp(−t/τ₁) − B₂ × exp(−t/τ₂)], with A = −0.033, B₁ = 0.62, B₂ = 0.22, τ₁ = 75.8 μsec, and τ₂ = 25.1 μsec. (B) The residuals of the fit. (A) The residuals of a single-exponential fit.

Figure 5.

(Bottom panel) A representative T-jump-induced relaxation trace of Mutant-2 measured at 1632 cm⁻¹. The T-jump was from 40.3°C to 49.0°C. The smooth line is the fit to the following single-exponential function: ΔOD(t) = A × [1 − exp(−t/τ)], with A = −0.019 and τ = 282 μsec. (Top panel) The residuals of the fit. (Note: The unresolved fast kinetic phase was subtracted.)

Figure 6.

(A) Arrhenius plot of the observed relaxation rate constants of Mutant-1. The symbols represent the slow, dominant rate constant (▵) as well as the fast rate constant (□), which is observed only at higher temperatures. (B) Arrhenius plot of the observed relaxation rate constant (▵) as well as folding (○) and unfolding (⋄) rate constants of Mutant-2. Solid lines are fits to Equations (4) and (5).

Figure 7.

Relative percentages of the slow kinetic phase of Mutant-1 corresponding to different final temperatures, obtained from experiment (○) and calculation (•).