1 research outputs found
Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy
The conformation
and dynamics of the unfolded state of ubiquitin
doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated
using single-molecule fluorescence spectroscopy. The line confocal
fluorescence detection system combined with the rapid sample flow
enabled the characterization of unfolded proteins at the improved
structural and temporal resolutions compared to the conventional single-molecule
methods. In the initial stage of the current investigation, however,
the single-molecule Förster resonance energy transfer (sm-FRET)
data of the labeled ubiquitin were flawed by artifacts caused by the
adsorption of samples to the surfaces of the fused-silica flow chip
and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl
phosphorylcholine polymer to the flow chip surface was found to suppress
the artifacts. The sm-FRET measurements based on the coated flow chip
demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin
at the native condition were narrowly distributed, which is comparable
to the probability density function (PDF) expected from the shot noise,
demonstrating the structural homogeneity of the native state. In contrast,
the histogram of the sm-FRET efficiencies of the unfolded ubiquitin
obtained at a time resolution of 100 μs was distributed significantly
more broadly than the PDF expected from the shot noise, demonstrating
the heterogeneity of the unfolded state conformation. The variety
of the sm-FRET efficiencies of the unfolded state remained even after
evaluating the moving average of traces with a window size of 1 ms,
suggesting that conformational averaging of the heterogeneous conformations
mostly occurs in the time domain slower than 1 ms. Local structural
heterogeneity around the labeled fluorophores was inferred as the
cause of the structural heterogeneity. The heterogeneity and slow
dynamics revealed by the line confocal tracking of sm-FRET might be
common properties of the unfolded proteins