Dual Illumination Enhances Transformation of an Engineered Green-to-Red Photoconvertible Fluorescent Protein

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes. We implemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins. With the improved H-bonding network and structural motions, the photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species.     

Fluorescence lifetimes in BO2 (A2Πu)

Fluorescence lifetimes and fluorescence quenching rates are reported for several vibronic levels in the A²Π_u electronic manifold of BO₂. The relationship of these results to earlier Hanle-effect measurements is discussed.     

Revealing Conformational Variants of Solution‐Phase Intrinsically Disordered Tau Protein at the Single‐Molecule Level

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution‐phase structural studies. We employed an anti‐Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time‐resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.