We developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing... Show moreWe developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing fluorophore ATTO655 was selected for labeling Azurin, a small blue mononuclear copper protein. Single molecule fluorescence correlation spectroscopy (FCS) of the fluorescently labeled Copper azurin in solution reveals how the position of the label in the 3-D structure of the protein affects the redox kinetics of the redox center as well as the label. Under certain redox conditions, we have been able to observe a microsecond dynamics for intramolecular ET reaction between the label and the metal center in azurin. Our results show that this FRET technique can be profitably used to study the enzyme activity of dye-labeled oxidoreductases. Show less
We study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double... Show moreWe study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double-stranded DNA molecules to access micro-second dynamics of single molecules, and reveal the influences of dye-dye interaction at short interdye distances on the observed FRET values. We use neutron-scattering techniques to examine the origin of solid-like structures suggested in previous reports and the influence of the thermal history. We find that crystal nucleation takes place in glycerol at temperatures very close to the glass transition temperature. This observation suggests that the thermal history of the glycerol sample needs to be controlled for studying dynamical heterogeneity in supercooled liquids. For the first time, we demonstrate gold nanorods as local viscosity reporter to study heterogeneity in supercooled liquids. Following rotational dynamics of individual gold nanorods in glycerol upon cooling below 226K, we start to observe deviations of local viscosity from the bulk viscosity of glycerol. Our observation suggests heterogeneity on relatively large length scale exists at surprisingly high temperatures. In the end, we demonstrate gold nanorods for enhancing fluorescence from single molecules and for fluorescence correlation spectroscopy at micromolar concentrations with single-molecule sensitivity. Show less
