Add like
Add dislike
Add to saved papers

Distance-Matched Tagging Sequence Optimizes Live-Cell Protein Labeling by a Biarsenical Fluorescent Reagent AsCy3_E.

ACS Omega 2018 Februrary 29
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 ( K d < 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.

Full text links

We have located links that may give you full text access.
Can't access the paper?
Try logging in through your university/institutional subscription. For a smoother one-click institutional access experience, please use our mobile app.

For the best experience, use the Read mobile app

Mobile app image

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app

All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.

By using this service, you agree to our terms of use and privacy policy.

Your Privacy Choices Toggle icon

You can now claim free CME credits for this literature searchClaim now

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app