Impact of In‐Cell and In‐Vitro Crowding on the Conformations and Dynamics of an Intrinsically Disordered Protein

Abstract

The conformations and dynamics of proteins can be influenced by crowding from the large concentrations of macromolecules within cells. Intrinsically disordered proteins (IDPs) exhibit chain compaction in crowded solutions in vitro, but no such effects were observed in cultured mammalian cells. Here, to increase intracellular crowding, we reduced the cell volume by hyperosmotic stress and used an IDP as a crowding sensor for in‐cell single‐molecule spectroscopy. In these more crowded cells, the IDP exhibits compaction, slower chain dynamics, and much slower translational diffusion, indicating a pronounced concentration and length‐scale dependence of crowding. In vitro, these effects cannot be reproduced with small but only with large polymeric crowders. The observations can be explained with polymer theory and depletion interactions and indicate that IDPs can diffuse much more efficiently through a crowded cytosol than a globular protein of similar dimensions.

Conformational Dynamics of Intrinsically Disordered Proteins Regulate Biomolecular Condensate Chemistry

“… 172 Translational diffusion of IDPs, which happens on the scale of micrometers, is even more slowed down than chain dynamics, for which the relevant length scale is in the nanometer regime. 113 Importantly, crowding might not have a significant impact on chain conformational sampling, as the amplitudes of motions have been reported to remain almost constant. 113 However, high protein concentrations inside droplets can cause a transition to the semidilute regime, where protein molecules penetrate each other’s hydrodynamic volumes, which can restrict chain flexibility.…”

“… 113 Importantly, crowding might not have a significant impact on chain conformational sampling, as the amplitudes of motions have been reported to remain almost constant. 113 However, high protein concentrations inside droplets can cause a transition to the semidilute regime, where protein molecules penetrate each other’s hydrodynamic volumes, which can restrict chain flexibility.…”

“…For translational diffusion, the relevant scale is in the micrometer range, above the characteristic length of intracellular crowders, whereas for the chain reorientation it is in the low nanometer regime, i.e., below this length. 113 To further probe the length scale effects on crowder viscosity and molecular diffusion, the small fluorophore Atto 532 (molecular weight MW = 0.9 kDa; hydrodynamic radius r h = 0.5 nm) and the folded protein β-glucuronidase (MW = 280 kDa; r h = 5.1 nm) were injected into crowded HeLa cells. Surprisingly, the relative diffusion time of ProTα (MW = 12 kDa; r h = 4.4 nm) in crowded cells was faster than for the folded protein β-glucuronidase of almost the same hydrodynamic radius and close to that of a very small molecule Atto 532.…”

“…(b) Relative diffusion times (τ d ) and (c) relative τ r values obtained in buffer, in HeLa cells cytosol without and with hyperosmotic stress. 113 The fence indicates the total range of τ r values, the white line indicates the median value, the black line indicates the mean value, and the colored box indicates the range between the first and the third quartile. The statistical significance of differences between mean values was verified by the Kolmogorow–Smirnow test (** P < 0.01, *** P < 0.001).…”

“…The statistical significance of differences between mean values was verified by the Kolmogorow–Smirnow test (** P < 0.01, *** P < 0.001). Adapted with permission from ref ( 113 ). Copyright 2021 John Wiley & Sons.…”