Macromolecular crowding in cells influences a myriad of biochemical processes, such as protein folding, diffusion, and equilibria. Previously, we have studied the environmental sensitivity of various genetically encoded donor-linker-acceptor constructs to macromolecular crowding as a function of Ficoll-70-a crowding agent-concentration with various fluorescence techniques. Here, we investigate the environmental sensitivity of an mCerulean3-linker-mCitrine construct-E6G2-with macromolecular crowding agents of varying size and surface charge. E6G2 was tested in buffer solutions containing 0 to 300 g/L of crowding agents. We hypothesize that increasing crowding agent size will result in more excluded volume, pushing E6G2 together in a more compact conformation, decreasing donor-acceptor distance and increasing FRET efficiency. We also hypothesize that E6G2 will be more sensitive to crowding agent size, rather than charge, as we have previously demonstrated these sensors are nonsensitive to ionic strength in solutions of KCl. To test these hypotheses, we measured the fluorescence lifetime of E6G2 in solutions enriched in varying crowding agents. This enables FRET efficiency and subsequently the donor-acceptor distance to be quantified. The dynamic viscosity of the crowding agents was also measured to determine overlap concentration and for use in future diffusion studies. By studying the environmental sensitivity of donor-linker-acceptor constructs with various crowding agents, we fill gaps in how crowding can affect proteins in unique ways beyond the effects observed in previous studies using Ficoll-70. This understanding can help in conceptualizing how crowding influences protein dynamics in more complex environments, such as in living cells.
Olson et al. (Sun,) studied this question.