Single-Molecule Analysis of Cytochrome <i>c</i> Folding by Monitoring the Lifetime of an Attached Fluorescent Probe
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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 <i>c</i> molecules. In particular, measurements
of the fluorescence lifetimes of individual cytochrome <i>c</i> 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