Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy on the Folding Mechanism of B Domain of Protein A

Abstract

Molecular-level description of the folding process of proteins is of fundamental importance for understanding how proteins acquire their unique conformations to show various biological functions. Elucidation of the folding mechanism requires quantitative analysis with a high time-resolution. To this aim, we recently developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS)1,2. This method enables us to examine the microsecond conformational dynamics of proteins at the single-molecule level, through the analysis of the fluorescence lifetime correlation. In this study, we applied 2D FLCS to study the folding mechanism of B domain of protein A (BdpA). This small protein was believed to show a two-state folding process in which the native state unfolds without exhibiting any intermediate states. However, a recent single-molecule study suggested that the folding mechanism of BdpA is not so simple3. To quantitatively analyze the complex folding process of BdpA, we performed 2D FLCS for two FRET mutants of BdpA. The results showed that the conformations of both the native and unfolded states are highly heterogeneous and that the conformational dynamics within each conformational ensemble occurs on a time scale shorter than ten microseconds. Furthermore, our results indicated that the conformational distribution in the native and unfolded ensembles gradually change with the change of the denaturant concentration.[1] Ishii and Tahara, 2013. J. Phys.Chem. B. 117, 11414-11422.[2] Ishii and Tahara, 2013. J. Phys.Chem. B. 117, 11423-11432.[3] Oikawa et al., 2013. Sci. Rep. 3, 2151.