Protein-based therapeutics are among the most precise and effective drugs on the market, yet their relatively short shelf-lives in aqueous solution, along with the high costs of refrigerated transport and storage, hinder their widespread use. To increase their stability and mitigate challenges associated with the so-called “cold chain,” many biologics and industrial enzymes are dehydrated in the presence of protective molecules known as excipients. Yet, despite decades of research, our understanding of how excipients protect proteins from dehydration is still limited, due in part to the technological inability to observe how water removal affects local protein structure. It is now possible to obtain residue-level information on dehydrated protein structures via liquid-observed vapor exchange (LOVE) NMR, a solution NMR technique that quantifies the extent of hydrogen–deuterium exchange between unprotected amide protons of a dehydrated protein and D2O vapor. In this poster, we describe the workflow and output of LOVE NMR, as well as how we applied it to globular proteins GB1 and CI2 to uncover the impact of mutation and excipients on the extent of dehydration-induced unfolding.
