Structural changes in bacteriorhodopsin during in vitro refolding from a partially denatured state

Abstract

We report on the formation of the secondary and tertiary structure of bacteriorhodopsin during its in vitro refolding from an SDS-denatured state. We used the mobility of single spin labels in seven samples, attached at various locations to six of the seven helical segments to engineered cysteine residues, to follow coil-to-helix formation. Distance measurements obtained by spin dipolar quenching in six samples labeled at either the cytoplasmic or extracellular ends of pairs of helices revealed the time dependence of the recovery of the transmembrane helical bundle. The secondary structure in the majority of the helical segments refolds with a time constant of <100-140 ms. Recovery of the tertiary structure is achieved by sequential association of the helices and occurs in at least three distinct steps with time constants of 1), well below 1 s; 2), 3-4 s; and 3), 60-130 s (the latter depending on the helical pair). The slowest of these processes occurs in concert with recovery of the retinal chromophore.

Figure 1

Topology diagram showing the locations of the designed cysteine mutations and labels in BR. The single mutants are shown as open circles and the double mutants are shown as shaded symbols. The residue locations for each pair of double mutants is represented with the same shaded symbol. The horizontal double lines approximate the membrane surfaces.

Figure 2

ESR spectrum of single-labeled denatured (thin lines) and regenerated (bold lines) states of F42CR1 (A), G63CR1 (B), A103CR1 (C), M163CR1 (D), V167CR1 (E), L201CR1 (F), and I222CR1 (G) BR samples, showing the difference in mobility of the spin label between the states. The spectra are normalized for equal numbers of spins in the D- and R-states. (H) Spectra of Cys+R1 in SDS micelles (thin line) and DMPC/CHAPS micelles (bold line).

Figure 3

ESR mobility change kinetics measured at 3341 G for F42CR1 (A), G63CR1 (B), A103CR1 (C), and M163CR1 (D). The intensity change observed for F42CR1 and A103CR1 is ∼5% of the expected change (magnitudes shown in the insets). The dotted horizontal line in black represents the signal intensity at time zero of the measurement. The arrows point to the time of actuation of the stop-flow pump, which initiates mixing and thus refolding.

Figure 4

ESR mobility change kinetics measured at 3341 G for V167CR1 (A), L201CR1 (B), I222CR1 (C), and control with l-cysteine attached to MTSL spin label in regenerating buffer mixed with SDS micelles (D). The dotted horizontal line in black represents the signal intensity at time zero of the measurement. The arrows point to the time of actuation of the stop-flow pump, which initiates mixing and thus refolding.

Figure 5

ESR dipolar interaction change kinetics measured at 3341 G for F42CR1/V167CR1 (A), F42CR1/I222CR1 (B), A103CR1/M163CR1 (C), V167CR1/I222CR1 (D), G63CR1/L201CR1 (E), and A126CR1/L201CR1 (F). The dotted horizontal line in black represents the signal intensity at time zero of the measurement.