SHEDDING LIGHT ON PROTEIN FOLDING DYNAMICS WITH HETEROGENEOUS UNFOLDED STATES
Spontaneous folding dynamics initiated by external electron transfer in equine heart cytochrome was monitored by time-resolved X-ray scattering. The systematic kinetic and structural analyses for the time-resolved data reveal that various protein conformations in the unfolded state take complex folding pathways, resulting in stretched exponential kinetics.
Protein undergoes a folding pathway to find a three-dimensional structure that expresses its biological function in a living cell. From vast theoretical studies, the funnel-like free-energy landscape for naturally avoiding Levinthal s paradox has been recognised as a key scheme for explaining the internal energies and conformational heterogeneities in protein folding . Despite numerous experimental efforts, however, studies connecting protein folding and the conformational heterogeneity in the funnel- like energy landscape have been rare even for small model proteins. Thus, directly observing the evolution of such structural heterogeneity is highly desirable for an in-depth understanding of protein folding at a molecular level.
To achieve this goal, time-resolved X-ray solution scattering (TRXSS), also known as time-resolved X-ray liquidography (TRXL), was performed at beamline ID09 to monitor the real-time evolution of unfolded equine heart cytochrome c (cyt-c) during folding. Cyt-c is a small metalloprotein with 104 amino acids and a heme group that is covalently bound to the protein backbone. Since the redox state of the heme acting as a cofactor is highly coupled with the folding free energy, it is possible to initiate spontaneous folding of cyt-c
under certain denaturing conditions. Namely, the protein is fully unfolded in its oxidised state while it is fully folded in its reduced state .
For the TRXSS study of cyt-c folding, the oxidised cyt-c with the unfolded conformation under a denaturing condition of 3.5 M GdnHCl was reduced by laser-induced electron transfer with nicotinamide adenine dinucleotide (NADH). The rapid photo-reduction subsequently initiated the folding process of cyt-c (Figure 48). This process was then tracked by X-ray probe pulses in the time domain ranging from early microseconds to late milliseconds (Figure 48). From the kinetic analysis of the time-resolved scattering data, it was possible to identify the stretched exponential kinetics in the transition of the early, unfolded intermediate to the folded state, which is sometimes referred to as strange kinetics . This feature observed in TRXSS is in stark contrast to the normal exponential behaviour reported from earlier spectroscopic studies, which interpreted it as sequential transitions of homogenously populated intermediates. To obtain structural information from the X-ray scattering curve, an ensemble-based structural analysis was implemented with the aid of a molecular dynamics (MD) simulation. It showed
Fig. 48: a) TRXSS data for redox-coupled folding of cyt-c. b) Schematic of photo- induced electron transfer from NADH to oxidised cyt-c via solvated electrons. c) Stretched exponential kinetics in the folding of cyt-c determined from the analysis of TRXSS data. The β value is an indicator of how far the kinetics deviates from a normal exponential function with β = 1. The observed folding has a β = 0.70 and a relaxation time of 185 ms.