A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges br

Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the sameprotein (intramolecular) or with a neighboring protein (intermolecu-lar), frequently due to changes in the cellular redox potential. Thereversible formation of non-native disulfides is intimately linked toalterations in protein function; while they can provide a mechanism toprotect against cysteine overoxidation, they are also involved in theearly stages of protein multimerization, a hallmark of several proteinaggregation diseases. Yet their identification using current proteinchemistry technology remains challenging, mainly because of theirfleeting reactivity. Here, we use single-molecule spectroscopy AFM andmolecular dynamics simulations to capture both intra- andintermolecular disulfide bonds in gamma D-crystallin, a cysteine-rich,structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as aconformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution