5FHV

Crystal structure of mCherry after reaction with 2-mercaptoethanol

Summary for 5FHV

• Entry DOI: 10.2210/pdb5fhv/pdb
• Related: 2H5Q
• Descriptor: mCherry-BME, TRIETHYLENE GLYCOL, BETA-MERCAPTOETHANOL, ... (5 entities in total)
• Functional Keywords: fluorescent protein, 2-mercaptoethanol, michael addition
• Biological source: Discosoma sp.
• Total number of polymer chains: 1
• Total formula weight: 31156.14

Authors

De Zitter, E.,Dedecker, P.,Van Meervelt, L. (deposition date: 2015-12-22, release date: 2017-01-11, Last modification date: 2024-01-10)

Primary citation

Cloin, B.M.C.,De Zitter, E.,Salas, D.,Gielen, V.,Folkers, G.E.,Mikhaylova, M.,Bergeler, M.,Krajnik, B.,Harvey, J.,Hoogenraad, C.C.,Van Meervelt, L.,Dedecker, P.,Kapitein, L.C.
Efficient switching of mCherry fluorescence using chemical caging.
Proc. Natl. Acad. Sci. U.S.A., 114:7013-7018, 2017

PubMed Abstract: Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

PubMed: 28630286
DOI: 10.1073/pnas.1617280114

Experimental method

X-RAY DIFFRACTION (1.55 Å)