A new model successfully predicts the size-dependent effects of crowding by polyethylene glycols, dextrans, and Ficolls on protein and protein-complex stability. However, cells are crowded with globular proteins, unlike these much-less-structured polymers. To test the model under more physiologically-relevant conditions, a series of AI-designed protein crowders with similar surface chemistry but increasing size were designed and produced. We assessed their stability over the temperature range needed to quantify the van 't Hoff enthalpy of the model protein SH3 when crowded by these novel proteins. Specifically, we have acquired circular dichroism spectra from room temperature to 98 °C and applied a two-state model to assess their stability. The designed proteins are highly thermal stable across this temperature range. We are now quantifying the effect of the designed protein crowders on the stability of SH3 by using 19 F NMR spectroscopy as a function of temperature. We observe that the free energy of SH3 unfolding increases with increasing crowder size, and the increases are enthalpically driven.
Davis et al. (Sun,) studied this question.