Understanding the fundamental forces such as hydrophobic interactions in a crowded intracellular environment is necessary to comprehensively decipher the mechanisms of protein folding and biomolecular self-assemblies. The widely accepted entropic depletion view of crowding effects primarily attributes biomolecular compaction to the solvent excluded volume effects exerted by the “inert” crowders, neglecting their soft interactions with the biomolecule. In this study, we examine the effects of chemical nature and soft attractive energy of crowders on the water-mediated hydrophobic interaction between two non-polar neopentane solutes using molecular dynamics simulations. The crowded environment is modeled using dipeptides composed of polar and non-polar amino acids of varying sizes. The results show that amongst the non-polar crowders, Leu2 strengthens the hydrophobic interactions significantly, whereas the polar and small-sized non-polar crowders do not show significant strengthening. Distinct underlying thermodynamic driving forces are illustrated where the small-sized crowders drive hydrophobic interaction via a classic entropic depletion effect and the bulky crowders strengthen it by preferential interaction with the solute. A crossover from energy-stabilized solvent-separated pair to entropy-stabilized contact pair state is observed in the case of bulky non-polar (Leu2) and polar (Lys2) crowders. The influence of solute–crowder energy in affecting the dehydration energy penalty is found to be crucial for determining the neopentane association. The findings demonstrate that along with the entropic (size) effects, the energetic effects also play a crucial role in determining hydrophobic association. The results can be extended and have implications in understanding the impact of protein crowding with varying chemistry in modulating the protein free energy landscapes.
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