Microsecond Conformational Dynamics of Cytochrome C Revealed by Two-Dimentional Fluorescence Lifetime Correlation Spectroscopy

Abstract

Characterization of the folding process is a long-standing central issue in protein science. Single-molecule spectroscopy, especially that in combination with fluorescence resonance energy transfer (FRET), has been utilized as a powerful tool to explore the conformational heterogeneity of proteins and its transition dynamics on the sub-millisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still challenging. Elucidation of protein dynamics in the microsecond region is very crucial to understand elementary processes of not only folding but also various biological functions of proteins. We recently developed a new single-molecule technique to quantitatively examine the microsecond dynamics of biomolecules based on fluorescence lifetime correlation analysis. This method, two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) [1,2], was applied to the spontaneous conformational transition of cytochrome c (cyt c) in this study. One fluorophore, Alexa546, was covalently attached as a FRET donor to the single free cysteine residue of cyt c located in the C-terminal region. The temporal change in the donor fluorescence lifetime due to FRET between the donor and heme was then analyzed to evaluate the conformational transition dynamics of cyt c. We show that 2D FLCS reveals diverse conformers of cyt c and provides unambiguous information about their microsecond transition dynamics. This work demonstrates the high capability of 2D FLCS to elucidate the complex conformational transition dynamics of proteins.