Cell permeable biarsenical
fluorescent dyes built around a cyanine
scaffold (AsCy3) create the ability to monitor the structural dynamics
of tagged proteins in living cells. To extend the capability of this
photostable and bright biarsenical probe to site-specifically label
cellular proteins, we have compared the ability of AsCy3 to label
two different tagging sequences (i.e., CCKAEAACC and CCKAEAAKAEAAKCC),
which were separately engineered onto enhanced green fluorescent proteins
(EGFPs) and expressed in Escherichia coli. The cysteine pairs within the shorter protein tag (i.e., Cy3TAG)
are designed to specifically match the 14.5 Å interarsenic atomic
separation within AsCy3, whereas the longer protein tag (Cy3TAG+6)
was identified using a peptide screening approach and reported to
enhance the binding affinity and brightness. We report that AsCy3
binds both the tagged proteins with similar high affinities (Kd < 1 μM) under both in vivo labeling
conditions and following isolation and labeling of the tagged EGFP
protein. Greater experimental reproducibility and substantially larger
AsCy3 labeling stoichiometries are observed under in vivo conditions
using the shorter Cy3TAG in comparison to the Cy3TAG+6. These results
suggest that the use of the distance-matched and conformationally
restricted Cy3TAG avoids nonspecific protein interactions, thereby
enabling routine measurements of protein localization and conformational
dynamics in living cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.