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Shape shifting linked to disease: A single‐molecule fluorescence technique was used to probe structures of an intrinsically disordered brain protein. A mutation was found to tilt the coupled binding–folding energy landscape of the protein and inhibited switching between induced ordered structures (see picture). The observations provide fundamental insight into the molecular basis of Parkinson's disease.

Krankhafte Verformungen: Eine Einzelmolekül‐Fluoreszenztechnik wurde bei Strukturuntersuchungen an einem intrinsisch fehlgeordneten Gehirnprotein angewendet. Bei einer Mutation wurde festgestellt, dass sie die gekoppelte Bindungs‐ und Faltungsenergie des Proteins veränderte und das Schalten zwischen induzierten geordneten Strukturen verhinderte (siehe Bild). Die Beobachtungen liefern grundlegende Informationen zu molekularen Vorgängen bei der Parkinson‐Krankheit.

Single-molecule fluorescence is widely used to study conformational 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 two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links 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 global smFRET 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.

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