Efficient switching of mCherry fluorescence using chemical caging

TY - JOUR

T1 - Efficient switching of mCherry fluorescence using chemical caging

AU - Cloin, Bas M.C.

AU - De Zitter, Elke

AU - Salas, Desiree

AU - Gielen, Vincent

AU - Folkers, Gert E

AU - Mikhaylova, Marina

AU - Bergeler, Maike

AU - Krajnik, Bartosz

AU - Harvey, Jeremy

AU - Hoogenraad, Casper C

AU - Van Meervelt, Luc

AU - Dedecker, Peter

AU - Kapitein, Lukas C.

PY - 2017/7/3

Y1 - 2017/7/3

N2 - 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.

AB - 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.

KW - Journal Article

U2 - 10.1073/pnas.1617280114

DO - 10.1073/pnas.1617280114

M3 - SCORING: Journal article

C2 - 28630286

VL - 114

SP - 7013

EP - 7018

JO - P NATL ACAD SCI USA

JF - P NATL ACAD SCI USA

SN - 0027-8424

IS - 27

ER -