Single-Molecule Studies Reveal That Water Starts to Protect Proteins Even at the ppm Level

A consensus established by extensive thermodynamic and structural studies of proteins and DNA is that water acts as an indispensable structural cofactor rather than a passive solvent for biomolecules. Nevertheless, it remains unclear whether trace water still exerts a stabilizing effect on proteins under ultradry conditions. Nonpolar organic solvents that spontaneously absorbed trace water provide an ideal platform to investigate this issue. Herein, atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) was employed to investigate the mechanical behaviors of two model polyproteins, (GB1)8 and (I27)8, in nonpolar organic solvents with varying water contents. It was found that even at the ppm level, water protects protein stability and acts as a molecular switch: polyproteins fully denature at water contents ≤80 ppm (nonane and tetradecane), while metastable folded fragments can be retained at water contents ≥95 ppm (tetrachloroethane (TCE) and octane) with a characteristic unfolding force of approximately 100 pN. Force-free molecular dynamics (MD) simulations of a smaller (I27)2 model in TCE further suggested that trace water accumulated preferentially around the protein, reorganizing the local solvation environment, thereby serving as a stabilizer. Based on the SMFS results, the water-content range of 80-95 ppm was identified as a critical threshold, across which polyproteins toggle between fully denatured and partially stabilized states. This work reveals the protective effect of trace water on proteins, quantifies its stabilizing capacity down to the ppm level, and offers insights for protein storage and astrobiology research.