Trimethylamine N-oxide (TMAO) and urea are two important osmolytes with their main significance to the biophysical field being in how they uniquely interact with proteins. Urea is a strong protein destabilizing agent, whereas TMAO is known to counteract urea's deleterious effects. The exact mechanisms by which TMAO stabilizes and urea destabilizes folded proteins continue to be debated in the literature. Although recent evidence has suggested that urea binds directly to amino acid side chains to make protein folding less thermodynamically favored, it has also been suggested that urea acts indirectly to denature proteins by destabilizing the surrounding hydrogen bonding water networks. Here, we elucidate the molecular level mechanism of TMAO's unique ability to counteract urea's destabilizing nature by comparing Raman spectroscopic frequency shifts to the results of electronic structure calculations of microsolvated molecular clusters. Experimental and computational data suggest that the addition of TMAO into an aqueous solution of urea induces blue shifts in urea's H-N-H symmetric bending modes, which is evidence for direct interactions between the two cosolvents.

The effects of trimethylamine N‐oxide (TMAO) on guanidinium chloride and hydrogen‐bonded networks of water are explored in this joint Raman spectroscopic and quantum chemical study. Both TMAO and guanidinium are osmolytes that affect the stability of proteins, as TMAO is known to stabilize and counteract the destabilizing effects of guanidinium. While guanidinium is very similar in chemical structure to urea, the exact mechanisms of the molecular interactions between guanidinium, TMAO, and proteins continue to be investigated. Herein, we use Raman spectroscopy to elucidate the physical interactions between TMAO and guanidinium in aqueous solutions to better understand how these important osmolytes interact with each other and affect adjacent hydrogen‐bonding networks of water. Comparing experiment to theory yields good agreement and allows for the identification and tracking of different vibrational modes. It was determined that adding TMAO into an aqueous solution of guanidinium induces a blue shift (shift to higher energy) in guanidinium's H‐N‐H bending modes, which is indicative of direct interactions between the two osmolytes and similar to the earlier results observed for TMAO interacting with urea.