Folding energy landscape of cytochrome cb562

Abstract

Cytochrome cb(562) is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb(562) refolding triggered using an ultrafast continuous-flow mixer (150 micros dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59-heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b(562) folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.

Fig. 1.

GuHCl-induced unfolding of cyt cb₅₆₂ (pH 5.0, 50 mM NaOAc) probed by using Trp fluorescence decay. (A) Normalized Trp-59 fluorescence decay curves of cyt cb₅₆₂. For clarity, only 6 of the 19 observed decays are displayed; 0 M (black), 2.0 M (red), 4.0 M (blue), 4.5 M (green), 5.4 M (yellow), and 6.9 M (gray). (B) Distributions of Trp-59–heme center-to-center distances [P(r_DA)] extracted from maximum entropy analyses of the TRFET data. The area of each bar reflects the probability amplitude over the corresponding distance range. (C) Population changes of each component as a function of the GuHCl concentration (native, blue; short, green; intermediate, red; and extended, black). Solid lines are two-state fitting curves with [GuHCl]_1/2 = 4.2 M and m = 7.5 kJ mol⁻¹ M⁻¹.

Fig. 2.

Folding kinetics of cyt cb₅₆₂ triggered by GuHCl jump from 6.0 M to 1.0 M in a CTF mixer (pH 5.0, 18 °C) probed by Trp-59 fluorescence. (A) TRFET data (8 of the 112 measured decays are displayed): 0 (unfolded in 6.0 M GuHCl, gray), 150 μs (red), 430 μs (blue), 1.25 ms (green), 3.41 ms (orange), 6.50 ms (purple), 7.70 ms (yellow), and >30 min (native state in 1.0 M GuHCl, black) after the initiation of folding reaction. (B) Integrated Trp-59 fluorescence intensity (Φ_fl) as a function of folding time. The 112 original data points are logarithmically smoothed for clarity. Solid line is a fit of the full dataset to a single exponential function with a rate constant of 2.6 (±0.2) × 10² s⁻¹.

Fig. 3.

Distributions of P(r_DA) for refolding of cyt cb₅₆₂. The P(r_DA) distributions are extracted from maximum entropy analyses of the FET kinetics data. Folding was triggered by GuHCl jump from 6.0 M to 1.0 M in a CTF mixer (pH 5.0, 18 °C). For clarity, only 8 of the 112 observed decays are displayed: (A) unfolded in 6.0 M GuHCl; (B) 150 μs after the initiation of folding reaction; (C) 430 μs; (D) 1.25 ms; (E) 3.41 ms; (F) 6.50 ms; (G) 7.70 ms; (H) >30 min (native state in 1.0 M GuHCl).

Fig. 4.

Folding kinetics of cyt cb₅₆₂ (112 original data points are logarithmically smoothed for clarity). (A) Mean distance between tryptophan and heme (M₁) as a function of folding time. The solid line is a fit of a single exponential function: rate constant of 2.6 (± 0.2) × 10² s⁻¹. (B) Time course of the second moment (M₂). The solid line is a fit of a single exponential function: rate constant of 2.4 (±0.3) × 10² s⁻¹. (C) Time course of the variance (v). The solid line is a fit of a double exponential function: rate constants of 5.1 × 10² and 2.6 × 10² s⁻¹.