Revisiting the folding kinetics of bacteriorhodopsin

Abstract

The elucidation of the physical principles that govern the folding and stability of membrane proteins is one of the greatest challenges in protein science. Several insights into the folding of α-helical membrane proteins have come from the investigation of the conformational equilibrium of H. halobium bacteriorhodopsin (bR) in mixed micelles using SDS as a denaturant. In an effort to confirm that folded bR and SDS-denatured bR reach the same conformational equilibrium, we found that bR folding is significantly slower than has been previously known. Interrogation of the effect of the experimental variables on folding kinetics reveals that the rate of folding is dependent not only on the mole fraction of SDS but also on the molar concentrations of mixed micelle components, a variable that was not controlled in the previous study of bR folding kinetics. Moreover, when the molar concentrations of mixed micelle components are fixed at the concentrations commonly employed for bR equilibrium studies, conformational relaxation in the transition zone is slower than hydrolysis of the retinal Schiff base. As a result, the conformational equilibrium between folded bR and SDS-denatured bR cannot be achieved under the conventional condition. Our finding suggests that the molar concentrations of mixed micelle components are important experimental variables in the investigation of the kinetics and thermodynamics of bR folding and should be accounted for to ensure the accurate assessment of the conformational equilibrium of bR without the interference of retinal hydrolysis.

Figure 1

Folding of bR monitored by pulse proteolysis. The amount of refolded bR was determined by pulse proteolysis after equilibration of unfolded bR at varying X_SDS for 3 min (○). The f_N values were determined by dividing the band intensities of remaining intact bR after pulse proteolysis by the intensity of the folded bR treated with pulse proteolysis. For comparison, the f_N values determined by pulse proteolysis after equilibration of folded bR at varying X_SDS (•) are shown. The f_N values determined after equilibration of folded bR were fit to a two-state equilibrium model (solid line).

Figure 2

The effect of DMPC and CHAPSO concentrations on folding and unfolding kinetics of bR. bR unfolding and refolding were monitored by the absorbance at 560 nm (A₅₆₀) in buffers containing the same X_SDS but different molar concentrations of DMPC and CHAPSO. A: bR was refolded at 0.50 X_SDS in buffers containing 15 mM DMPC and 16 mM CHAPSO (15/16 buffer, dashed line) or 29 mM DMPC and 31 mM CHAPSO (29/31 buffer, solid line). B: bR was unfolded at 0.81 X_SDS in 15/16 buffer (dashed line) and in 29/31 buffer (solid line). C: The rate constants for bR refolding at 0.50 X_SDS (•) and for bR unfolding at 0.81 X_SDS (○) were determined in varying molar concentrations of DMPC and CHAPSO. The ratio of the molar concentration of DMPC to that of CHAPSO was fixed to 15:16. The plots of the logarithms of the rate constants versus X_SDS are fitted to a linear function.

Figure 3

The kinetics of conformational relaxation of bR at varying X_SDS with fixed concentrations of DMPC and CHAPSO. The natural logarithm of the observed rate constant (k_obs) for unfolding (⋄) and refolding (○) in 15/16 buffer as well as the natural logarithm of k_obs for unfolding (♦) and refolding (•) in 29/31 buffer are plotted against X_SDS. The rate constant for the hydrolysis of the retinal Schiff base (k_h) is indicated for comparison (dashed line). The fitting of lnk_obs versus X_SDS reported by Curnow and Booth is also shown for comparison (solid line).

Figure 4

The formation of bacterioopsin during bR folding. Absorbance spectra were taken at several time points following the initiation of refolding in 15/16 buffer under conditions in which bR folding is similar to the rate of retinal hydrolysis (0.65 X_SDS) (A) and under conditions in which bR folding is significantly faster than the rate of retinal hydrolysis (0.40 X_SDS) (B). The arrow indicates the order of the spectra taken over time during refolding. Vertical lines indicate the absorbance maxima of native bR (bR_F, 560 nm), SDS-denatured bR (bR_U, 440 nm), and bacterioopsin + free retinal (bO, 390 nm).

Figure 5

Relaxation under higher concentrations of DMPC and CHAPSO. The folding kinetics of bR in 45 mM DMPC and 48 mM CHAPSO (45/48) is predicted based on the folding kinetics in 29/31 buffer shown in Figure 3 and the effects of the concentrations of DMPC and CHAPSO on the folding kinetics of bR shown in Figure 2(C). Only the linear portions of the folding (X_SDS = 0.40–0.60, •) and unfolding (X_SDS = 0.74–0.81, ○) kinetics data in 29/31 buffer are used for the analysis. The kinetic C_m in 45 mM DMPC and 48 mM CHAPSO is estimated to be 0.79 X_SDS. Also, the relaxation rate constant at the C_m value in 45 mM DMPC and 48 mM CHAPSO is estimated to be 0.030 s⁻¹, which 30-fold greater than retinal hydrolysis constant (k_h = 0.0010 s⁻¹, dashed line).