Pump-Probe X-ray Solution Scattering Reveals Accelerated Folding of Cytochrome c Upon Suppression of Misligation.

Generally, a protein molecule should form its native structure to perform its biological function in the living cell. However, the failure of proper folding, i.e. misfolding, occurs occasionally and this causes aggregation and/or degradation of the protein, which is potentially linked to protein mis-folding diseases, e.g. prion disease and Parkinson's disease. Therefore, it is critical to understand the misfolding mechanism , as well as protein folding, in order to develop preventative procedures. Among the numerous available proteins, horse heart cyto-chrome c (Cyt c) is one of the most intensively studied model system (Figure 1(a)). 2 Cyt c has a fully unfolded structure under a certain concentration of denaturant, and external ligands such as carbon monoxide (CO) are easily bound to the iron in the heme group. Many previous experiments have been conducted in order to examine the folding mechanism of Cyt c using the unfolded Cyt c bound with CO. An optical pump pulse has been used to initiate folding by dissociating CO from the heme group and its time-dependent processes have been probed using various spectroscopic techniques. Although the existing studies have proposed the possible kinetic models for the folding of Cyt c, a unified kinetic model has not yet been established due to the complexities that are caused by the misligation between histidine 26/33 (His26/33) and iron. This abnormal interplay disturbs the binding of methionine 80 (Met80) to the iron and may lead to misfolding. In this study, we explore the effect of misligation on the folding pathway of Cyt c using pump-probe (time-resolved) X-ray solution scattering. 8-10 We employ imidazole as an inhibitor that blocks the misligation. Imidazole has a similar structure to that of histidine (Figure 1(a)); due to this structural similarity, imidazole can transiently bind to iron. 11 This interaction can remove the complex ligand exchange process of misligated residues such as histidine 26 or 33. It is expected that the addition of imidazole at 50 times greater content than that of Cyt c will suppress the misligation between the His26/33 and heme through the transient binding of imidazole to the heme. Figure 1(b) presents two sets of difference scattering curves (i.e. with and without imidazole), ΔS(q, t), which are obtained by subtracting the laser-off scattering curve (reference) measured at –5 μs from the laser-on scattering curve at various positive time delays. (Refer to the Supporting Information for further experimental details.) A quantitative kinetic analysis …