Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation

Abstract

Single-molecule fluorescence is widely used to studyconformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than twodyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions andlinks to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more globalsmFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors.