Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications

We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the microenvironment and can be a “molecular ruler„ when combined with FRET. Utilization of fluorescence lifetime in 2D FLCS thus enables us to quantify the inhomogeneity of the system and the interconversion dynamics among different species with a higher time resolution than other single-molecule techniques. Recent applications of 2D FLCS to various biological systems demonstrate that 2D FLCS is a unique and promising tool to quantitatively analyze the microsecond conformational dynamics of macromolecules at the single-molecule level

Total Internal Reflection Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Fluorescence lifetime correlation analysis is becoming a powerful tool to understand the conformational heterogeneity of biomolecules and their dynamics with an unprecedented detection sensitivity and time resolution. However, its application to the study of biomembranes is very limited. Here, we report on two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) in combination with total internal reflection (TIR) microscopy (TIR 2D-FLCS). High depth resolution in TIR microscopy and species-specific correlation analysis in 2D FLCS give us the opportunity to selectively analyze molecules in or on a supported lipid bilayer, a model biomembrane formed on the glass surface. Feasibility experiments performed in this study clearly demonstrated that TIR 2D-FLCS has a potential to selectively analyze the diffusion and the conformational dynamics of proteins peripherally bound on the membrane in the presence of substantial amounts of unbound molecules in the bulk phase

Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Elucidating the protein folding mechanism is crucial to understand how proteins acquire their unique structures to realize various biological functions. With this aim, the folding/unfolding of small globular proteins has been extensively studied. Interestingly, recent studies have revealed that even such small proteins represent considerably complex processes. In this study, we examined the folding/unfolding process of a small α-helical protein, the B domain of protein A (BdpA), at equilibrium using two-dimensional fluorescence lifetime correlation spectroscopy with 10 μs time resolution. The results showed that although the BdpA is a two-state folder, both the native and unfolded states are highly heterogeneous and the conformational conversion within each ensemble occurs within 10 μs. Furthermore, it was shown that the average structures of both ensembles gradually change and become more elongated as the denaturant concentration increases. The analysis on two mutants suggested that fraying of the N-terminal helix is the origin of the inhomogeneity of the native state. Because the direct observation of the ensemble nature of the native state at the single-molecule level has not been reported, the data obtained in this study give new insights into complex conformational properties of small proteins