The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using th...

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribut...

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads ...

Applications of Fluorescence Resonance Energy Transfer (FRET) in biology have expanded tremendously the last 25 years. This technique has become a staple many biological and biophysical fields. FRET based chemical sensors biosensors can play an important role as biomarkers. Development new effective assay for application is emerging area research needing multidisciplinary approach from biologis...

We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert c...

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to ...

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, co...

Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET to imaging coexisting conformations of fibrone...

Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult ...

An optofluidic FRET (fluorescence resonance energy transfer) laser is formed by putting FRET pairs inside a microcavity acting as a gain medium. This integration of an optofluidic laser and the FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here, we investigated...