Using deeply trapped intermediates to map the cytochrome c folding landscape

Abstract

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome c. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal-ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome c.

Figure 1

Overlay of Fe-Cyt c and Co-Cyt c (blue) structures in the heme region.

Figure 2

Comparison of Co-Cyt c and Har-Co-Cyt c refolding kinetics measured by CD spectroscopy (228 nm). (a) Unmodified horse Co-Cyt c (red), horse Har-Co Cyt c (blue); (b) unmodified tuna Co-Cyt c (magenta), tuna Har-Co-Cyt c (green). Folding was initiated by manual dilution of a 4.0 M GuHCl solution to give a final denaturant concentration of 2.0 M at 25°C. The horse and tuna proteins were incubated in unfolding solutions for 10 min and 2 days, respectively.

Figure 3

Far-UV CD spectra of Co-Cyt c species observed during refolding in 0.4 M GuHCl at 7°C. (a) Horse Co-Cyt c: unfolded (red); t = 1 min (green); 10 min (cyan); 55 min (magenta); folded (blue) (b) tuna Co-Cyt c: unfolded (red); t = 1 min (green), 17 min (cyan); 48 min (magenta); folded (blue).

Figure 4

(Upper) Luminescence decay kinetics of DNS(Cys 102)-Co-Cyt c measured at various times after initiating protein refolding by denaturant dilution {[GuHCl <0.5 M, pH 7}. (Lower) Distributions of excited-state decay rate constants [P(k)] and DNS-Co distances [P(r)] extracted from DNS luminescence decay kinetics at the specified folding times.

Figure 5

Schematic representation of a two-dimensional cross section of the Cyt c folding energy landscape. The shallow energy minima correspond to nearly degenerate extended (E) and collapsed (C, C′) conformations. The collapsed structures on the left side of the global energy minimum (C) can surmount a ligand substitution barrier to reach the native (N) structure. This substitution barrier in Co-Cyt c (blue) is much higher than that of Fe-Cyt c (red). Collapsed peptides on the right side (C′) face a high topological barrier to formation of the native fold; this population must extend and collapse again to reach compact structures with lower barriers to folding.