Single-Molecule Analysis of Cytochrome c Folding by Monitoring the Lifetime of an Attached Fluorescent Probe

Abstract

Conformational dynamics of proteins are important for function. However, obtaining information about specific conformations is difficult for samples displaying heterogeneity. Here, time-resolved fluorescence resonance energy transfer is used to characterize the folding of single cytochrome c molecules. In particular, measurements of the fluorescence lifetimes of individual cytochrome c molecules labeled with a single dye that is quenched by energy transfer to the heme were used to monitor conformational transitions of the protein under partially denaturing conditions. These studies indicate significantly more conformational heterogeneity than has been described previously. Importantly, the use of a purified singly-labeled sample made a direct comparison to ensemble data possible. The distribution of lifetimes of single-proteins was compared to the distribution of lifetimes determined from analysis of ensemble lifetime fluorescence data. The results show broad agreement between single-molecule and ensemble data, with a similar range of lifetimes. However, the single-molecule data reveal greater conformational heterogeneity.

Keywords: change-point algorithm; confocal microscopy; protein folding; single-molecule fluorescence lifetime.

Figure 1

(a) Scheme for immobilization of K99C-AF488 to a PEG-coated quartz coverslip. The arrow between the AF488 and the heme illustrates how the proximity changes between the two chromophores as the protein unfolds. (b) Images of K99C-AF488 in the presence of various concentrations of denaturant. Data was collected using a 100 μs integration time per pixel. The horizontal dimension is approximately 10 μm.

Figure 2

Representative fluorescence intensity and lifetime time trace for an individual molecule of K99C-A488 at 2.0 M GuHCl. (a) 50-ms binned intensity data (gray) with the intensity reconstructed by the change point algorithm (red) showing six different intensity states (I₁ – I₆). (b) TCSPC histograms (red markers) and the MLE fits (black line) for extracting the lifetimes for the five dye fluorescence intensity states (τ₂ – τ₆) from (a), compared with the background intensity (τ₁). Photon counts for each lifetime state are depicted below the lifetime values. (c) Extracted fluorescence lifetime (blue) as a function of time overlaid onto the 50-ms binned intensity trace (gray).

Figure 3

(a) Distribution of SM fluorescence lifetime states of K99C-AF488 at various concentrations of GuHCl. (b) Ensemble TCSPC histograms of 150 nM K99C-AF488 in solution (red), compared with TCSPC histograms of photons collected from SMs (black). The SM photon histogram in (b) includes only the photons used to generate the histograms in (a). (c) MEMexp fits of the ensemble decay curves (red line in (b)).

Figure 4

Two-dimensional histograms of transitions between lifetime states in SM time traces of K99C-AF488 at 1.75, 2.00, 2.25 and 2.50 M GuHCl. The data are plotted in 0.2 ns bins for ease in presentation. Points appearing on the diagonal correspond to small (but non-zero) changes in lifetime that both fall within a given bin.