Smaller molecules crowd better: Crowder size dependence revealed by single-molecule FRET studies and depletion force modeling analysis.

The cell is an extremely crowded environment, which is known to have a profound impact on the thermodynamics, functionality, and conformational stability of biomolecules. Speculations from recent theoretical molecular dynamics studies suggest an intriguing size dependence to such purely entropic crowding effects, whereby small molecular weight crowders under constant enthalpy conditions are more effective than larger crowders on a per volume basis. If experimentally confirmed, this would be profoundly significant, as the cellular cytoplasm is also quite concentrated in smaller molecular weight solutes such as inorganic ions, amino acids, and various metabolites. The challenge is to perform such studies isolating entropic effects under isoenthalpic conditions. In this work, we first present results from single-molecule FRET spectroscopy (smFRET) on the molecular size-dependent crowding stabilization of a simple RNA tertiary motif (the GAAA tetraloop-tetraloop receptor), indeed providing evidence in support of the surprising notion in the crowding literature that "smaller is better." Specifically, systematic smFRET studies as a function of crowder solute size reveal that smaller molecules both significantly increase the RNA tertiary folding rate and, yet, simultaneously decrease the unfolding rate, predicting strongly size-dependent stabilization of RNA tertiary structures under crowded cellular conditions. The size dependence of these effects has been explored via systematic variation of crowder size over a broad range of molecular weights (90-3000 amu). Furthermore, corresponding temperature dependent studies indicate the systematic changes in the folding equilibrium to be predominantly entropic in origin, i.e., consistent with a fundamental picture of entropic molecular crowding without additional enthalpic interactions. Most importantly, all trends in the single-molecule crowding data can be quantitatively recapitulated by a simple analytic depletion force model, whereby excluded volume interactions represent the major thermodynamic driving force toward folding. Our study, thus, not only provides experimental evidence and theoretical support for small molecule crowding but also predicts further enhancement of crowding effects for even smaller molecules on a per volume basis.