Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation

Abstract

The Engrailed Homeodomain protein has the highest refolding and unfolding rate constants directly observed to date. Temperature jump relaxation measurements gave a refolding rate constant of 37,500 s(-1) in water at 25 degrees C, rising to 51,000 s(-1) around 42 degrees C. The unfolding rate constant was 1,100 s(-1) in water at 25 degrees C and 205,000 s(-1) at 63 degrees C. The unfolding half-life is extrapolated to be approximately 7.5 ns at 100 degrees C, which allows real-time molecular dynamics unfolding simulations to be tested on this system at a realistic temperature. Preliminary simulations did indeed conform to unfolding on this time scale. Further, similar transition states were observed in simulations at 100 degrees C and 225 degrees C, suggesting that high-temperature simulations provide results applicable to lower temperatures.

Figure 1

Equilibrium denaturation of En-HD. Reversible thermal denaturation of En-HD as followed by circular dichroism (CD) and differential scanning calorimetry (DSC).

Figure 2

Relaxation kinetics of En-HD. (A) Kinetic trace at 25°C and 2.6 M urea on a 24,000-V electrical discharge with a 50-nF capacitor (5° jump). Six traces were averaged (En-HD concentration was 49 μM). Fitting of the observed trace to a single exponential equation gives a relaxation rate of 10,300 ± 200 s⁻¹. (B) Reaction trace at 57°C and 0 M urea on a 30,000-V electrical discharge from a 10-nF capacitor (1° jump). Seventeen traces were averaged (En-HD concentration was 300 μM). Fitting of the observed trace to a single exponential equation gives a relaxation rate of 145,000 ± 500 s⁻¹.

Figure 3

Folding kinetics at increasing temperatures. (A) Relaxation rate constants (♦) are plotted in a logarithmic axis vs. temperature. Folding (k_f, ○) and unfolding (k_u, □) rate constants were extracted from k_obs as described in the text, plotted in an Eyring plot of ln(k_obs/T) vs. 1/T (K⁻¹) (not shown) and fitted individually to ln (k/T) = ln (k_B/h) − ΔG^‡/RT to estimate the half-times of reaction up to 100°C shown in B.

Figure 4

Time progression of the thermal denaturation of En-HD at neutral pH from MD simulations at 100 and 225°C. The early steps in unfolding to the transition state and the disruption of helix packing are illustrated at 100°C. The unfolding at 100°C is on the proper time scale, as estimated from experiment. However, as the process is accelerated by temperature, the conformational heterogeneity of the denatured state is better sampled at 225°C. The crystal structure (17) is given as the 0-ns conformation. All structures are colored according to the placement of native helical structure: helix I, residues 10–22, in red; helix II, residues 28–38, in green; and helix III, residues 42–55, in blue. Helical structure, as determined using the method of Kabsch and Sander (43), is illustrated by ribbons. Trp-48, the fluorescence probe, is shown in magenta. This residue becomes exposed to solvent at the transition state and remains exposed thereafter at both temperatures. Note the similarity between the transition states at different temperatures: the displayed transition state structures have an α-carbon rms deviation of 3.8 Å, which is lower than their rms deviations from the starting structure (3.9 and 5.3 Å for 100 and 225°C, respectively).