Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin unfolding.

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37 ± 3 Å at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol