Long-Term Observation of Fluorescence of Free Single Molecules To Explore Protein-Folding Energy Landscapes

Abstract

A method was developed to detect fluorescence intensity signals from single molecules diffusing freely in a capillary cell. A unique optical system based on a spherical mirror was designed to enable quantitative detection of the fluorescence intensity. Furthermore, "flow-and-stop" control of the sample can extend the observation time of single molecules to several seconds, which is more than 1000 times longer than the observation time available using a typical confocal method. We used this method to scrutinize the fluorescence time series of the labeled cytochrome c in the unfolded state. Time series analyses of the trajectories based on local equilibrium state analysis revealed dynamically differing substates on a millisecond time scale. This system presents a new avenue for experimental characterization of the protein-folding energy landscape.

Single Molecule Data Analysis: An Introduction

“…Statistical approaches have been invaluable in generating detailed mechanistic insight into realms previously inaccessible at every step along biology's central dogma [42,43]. They have also unveiled basic molecular mechanisms from noisy single molecule data that have given rise to detailed energy landscape [44][45][46][47][48][49][50] and kinetic scheme [31,[51][52][53][54][55] models. Furthermore, one's choice of analysis methods can deeply alter the interpretation of experiment [56,57].…”

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

“…Proteins in the cytochrome c (cyt c ) protein family have been studied extensively in terms of protein folding and stability . Met80 and His18 are coordinated to the heme iron in cyt c , creating a relatively high redox potential for electron transfer.…”

“…18,19 Proteins in the cytochrome c (cyt c) protein family have been studied extensively in terms of protein folding and stability. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] Met80 and His18 are coordinated to the heme iron in cyt c, creating a relatively high redox potential for electron transfer. The redox potential of M80A cyt c was lower than that of wildtype cyt c, due to the loss of the Met80-heme iron coordination bond, 36 whereas M80A cyt c exhibited unusual O 2 and CO binding properties.…”

Microsecond dynamics of an unfolded protein by a line confocal tracking of single molecule fluorescence

“…In typical SMFS experiments, fluorescence photons from both donor and acceptor fluorophores labeled to a sample are detected simultaneously, and are analyzed based on the Förster resonance energy transfer (FRET) mechanism to obtain the inter-fluorophore distance. The power of SMFS is typically exemplified in the characterization of the unfolded proteins at the single molecule level4567. The structural and dynamical properties of the unfolded proteins can be routinely obtained by using SMFS.…”