title: Rotamer-Restricted Fluorogenicity of the Bis-Arsenical ReAsH.
abstract: Fluorogenic dyes such as FlAsH and ReAsH are used widely to localize, monitor, and characterize proteins and their assemblies in live cells. These bis-arsenical dyes can become fluorescent when bound to a protein containing four proximal Cys thiols-a tetracysteine (Cys4) motif. Yet the mechanism by which bis-arsenicals become fluorescent upon binding a Cys4 motif is unknown, and this nescience limits more widespread application of this tool. Here we probe the origins of ReAsH fluorogenicity using both computation and experiment. Our results support a model in which ReAsH fluorescence depends on the relative orientation of the aryl chromophore and the appended arsenic chelate: the fluorescence is rotamer-restricted. Our results do not support a model in which fluorogenicity arises from the relief of ring strain. The calculations identify those As-aryl rotamers that support fluorescence and those that do not and correlate well with prior experiments. The rotamer-restricted model we propose is supported further by biophysical studies: the excited-state fluorescence lifetime of a complex between ReAsH and a protein bearing a high-affinity Cys4 motif is longer than that of ReAsH-EDT2, and the fluorescence intensity of ReAsH-EDT2 increases in solvents of increasing viscosity. By providing a higher resolution view of the structural basis for fluorogenicity, these results provide a clear strategy for the design of more selective bis-arsenicals and better-optimized protein targets, with a concomitant improvement in the ability to characterize previously invisible protein conformational changes and assemblies in live cells.
authors.forename: Allison S
authors.initials: AS
authors.lastname: Walker
authors.affiliation: Department of Chemistry & Biochemistry, Swarthmore College , 500 College Avenue, Swarthmore, Pennsylvania 19081, United States.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Rablen
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
mesh_terms: D001152:Arsenicals / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008962:Models, Theoretical; D010078:Oxazines / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014783:Viscosity
chemical_list: D001152:Arsenicals; D005456:Fluorescent Dyes; D010078:Oxazines; D011506:Proteins; C454115:ReAsH-EDT2 compound; D012996:Solutions; D013438:Sulfhydryl Compounds; D003545:Cysteine