Dynamic and heterogeneous cellular ionic strength plays an important role in protein folding, enzymatic activities, DNA binding, protein-protein interactions, and cell osmosis. We investigated the effect of environmental ionic strength on the sensitivity of mCerulean3-linker-mCitrine construct (or RD) using Förster resonance energy transfer (FRET) analysis. For these hetero-FRET studies on the RD biosensor, we modified an empirical homo-FRET model. We also developed a theoretical model to describe from basic principles the time-resolved fluorescence depolarization resulting from both rotational dynamics and hetero-FRET. Here, we also demonstrate experimental proof-of-principle using wavelength-dependent time-resolved fluorescence anisotropy of intact and cleaved RD biosensor as a model system at varying concentrations of KCl (0-500 mM). To test our theory, we excited the donor (mCerulean3) and detected the time-resolved fluorescence polarizations (parallel and perpendicular) simultaneously as a function of the detection wavelength. The time-resolved anisotropy of the mCerulean3 (donor) emission yields mainly the fluorescence depolarization due to rotational dynamics for both intact and cleaved RD as a function of ionic strength. In addition to estimating the hydrodynamic radii of RD, our results are used to assess the potential segmental mobility of the flexible, non-spherical hetero-FRET construct. In contrast, time-resolved anisotropy of mostly the mCitrine (acceptor) emission yields the fluorescence depolarization due both to the rotational dynamics and hetero-FRET dynamics as a function of ionic strength. Our time-resolved fluorescence depolarization results are compared with the traditional benchmark of time-resolved fluorescence technique on the same construct. Our theoretical and experimental studies establish time-resolved fluorescence depolarization (or anisotropy) as a powerful approach for hetero-FRET and protein-protein interaction studies in either controlled environments or living cells.
Goitia et al. (Sun,) studied this question.