Abstract
How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.
Highlights
How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science
Protein folding is a central problem for which conformational heterogeneity and structural fluctuation are extensively studied
In spite of the importance of microsecond dynamics of proteins, application of the conventional single-molecule fluorescence resonance energy transfer (smFRET) technique is limited to dynamics slower than B100 ms because of the difficulty in collecting a sufficient number of photons to evaluate the FRET efficiency in a short bin time[14]
Summary
How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. We report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. We report on a new single-molecule spectroscopic method, two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), to quantitatively elucidate the microsecond conformational dynamics of proteins[18,19,20]. This method is an extension of fluorescence correlation spectroscopy, in which the correlation of the fluorescence lifetime is detected. The fluorescence lifetime correlation reveals the highly heterogeneous nature of cyt c and clarifies a variety of conformational dynamics including the transitions between conformers on a timescale of several microseconds
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