Glassy Dynamics in the Folding Landscape of Cytochrome c Detected by Laser Photolysis

Abstract

Ferrocytochrome c liganded with CO refolds to a nativelike compact state, called the M state, where the non-native Fe2+-CO contact persists. The M state resembles the generic molten globule-like states and can be driven to the native state by pulsed laser photolysis of the CO ligand. The microsecond kinetics measured all across the guanidine hydrochloride unfolding transition of the protein produce a chevron plot with accentuated rollover in the folding limb, suggesting a glass transition of M en route to N. The rate of exit of the folding structure from the kinetic trap(s) limits the overall rate of folding of M to N. Sulfate-induced deceleration of the observed folding rate suggests that the folding structure indeed is transiently frozen in glassy traps. The results connect the post-transition features of the funnel paradigm.

Folding Mechanism of Reduced Cytochrome c: Equilibrium and Kinetic Properties in the Presence of Carbon Monoxide

“…The activation energy is similar to the value (23 kcal mol −1 ) previously reported for Fe 2+ cyt c -CO at pH 7.4 prepared by dilution from alkaline solution 59. We and others43 have consistently observed a small decrease in dissociation rate between 0 and 3 M GuHCl, but the origin of this effect is not clear at this time (possible explanations include ionic strength and/or viscosity effects in addition to local structure formation following CO dissociation).…”

“…The dramatic stabilization of the protein upon reduction of the heme is not fully understood, but appears to be due to a combination of electrostatic effects (the reduced heme is electrically neutral while the oxidized heme carries a net charge of +1), differential affinity of the heme iron for the axial ligands between the native and unfolded conformations in both oxidation states, and dynamic/entropic contributions. The fact that only non-native forms of Fe 2+ cyt c bind a CO ligand with high affinity to form a photolabile ligand complex has opened unique opportunities for manipulating the conformational transitions of the protein and probing its dynamics on the µs to ms time scale 12,20,21,23,25,27,42,43. In their initial equilibrium characterization of Fe 2+ cyt c in the presence of CO, Jones et al12 already noticed that this system does not undergo a simple two-state unfolding transition.…”

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

“…There is accumulation of the molten globule-like state, called the B-state, of cyt-c under highly alkaline salt-containing aqueous media. 175,176 The B-state has a compact and largely mobile molecular state containing native-like secondary structure and hydrodynamic radius, but no rigid tertiary structure. The B-state is more stable than the A-state of cyt-c as it is braced by native-like structural order, modest dynamics, and a compact molecular organization.…”

Viscosity-dependent structural fluctuation of the M80-containing Ω-loop of horse ferrocytochrome c

“…The protein chain refolds trapping the CO molecule which is still bonded to the heme iron. Cyt c with the trapped CO (called NCO-state) is structurally native-like [59,60] and it represents a long-lived late kinetic intermediate [61,65,66,67]. Folding is blocked as long as the NCO-state stays frozen in the kinetic trap.…”

“…In this work, we have examined the influence of solvent viscosity on the structural fluctuation of presumably the M80-containing X-loop by measuring the rate of thermally-driven CO-dissociation from the natively folded carbonmonoxycytochrome c (NCO-state). Because of its smaller size and the content of the heme iron, carbonmonoxycytochrome c is a model in experimental protein dynamics studies [41,[57][58][59][60][61][62][63]. The CO-dissociation rate of NCOstate varies inversely with viscosity initially when the solvent viscosity is low (68.0 cP), but saturates at higher viscosity, indicating that the CO-dissociation from the NCO-state involves sequential stages that depend differently on solvent friction, i.e., solvent coupled and nonsolvent-coupled stages of the process [22,47].…”