Fig. 2: Spectroscopic characterization of increase in degrees of freedom in the interior of mCherry.

a The fluorophore group of mCherry (red) and the three Trp residues of mCherry (blue) pointing towards the interior of mCherry (green). b Time-resolved fluorescence anisotropy of 100 nM mCherry show inner degrees of freedom of fluorophore increase. Shown are the fluorescence anisotropy decays of mCherry in the absence of PEG 6,000 (black) and in the presence of 25% (red) and 50% (green) FVO of PEG 6,000, where only above 30% FVO a rapidly decaying component shows up, on top of the overall slowdown due to increase in viscosity and decrease in fluorescence lifetime. c–e Trp fluorescence spectra report on increase in FP interior degrees of freedom. The fluorescence spectra of the three Trp residues of 10 µM mCherry were recorded as a function of increasing FVO of PEG and are reported without normalization (c), and after normalization (d), and the area below the fluorescence spectra (e, black dots) and the wavelength at which fluorescence intensity is maximal (e, red dots) were reported. Note that the trend in the area below spectra was fit to a sigmoidal curve (e, dashed black line), with a best-fit midpoint at 23.3 ± 0.3% FVO of PEG.