Measuring of the quenching rate of Atto-655 by tryptophan

Intramolecular diffusion is an important, but often overlooked, property of intrinsically disordered proteins and plays an important role in folding, assembly, and aggregation. Fluorescence resonance energy transfer is used to observe reconfiguration over nanometer length scales while a close range quenching over Angstrom length scales provides a complementary view with different dynamics. There are several probe/quencher pairs that have been employed with varying levels of quantification of the quenching rate. Here, we measure the electron transfer quenching parameters of the fluorophore Atto-655 by tryptophan using fluorescence correlation spectroscopy. Measurements with varying concentrations of quencher with low diffusion yield a distance-dependent quenching rate. These parameters provide for a more quantitative analysis of measurements of intramolecular diffusion, particularly in crowded environments.