Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul;2(3):031203.
doi: 10.1117/1.NPh.2.3.031203. Epub 2015 Jun 19.

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

Yi Shen  1 , Tiffany Lai  1 , Robert E Campbell  1
Affiliations

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

Yi Shen et al. Neurophotonics. 2015 Jul.

Abstract

The inherent advantages of red-shifted fluorescent proteins and fluorescent protein-based biosensors for the study of signaling processes in neurons and other tissues have motivated the development of a plethora of new tools. Relative to green fluorescent proteins (GFPs) and other blue-shifted alternatives, red fluorescent proteins (RFPs) provide the inherent advantages of lower phototoxicity, lower autofluorescence, and deeper tissue penetration associated with longer wavelength excitation light. All other factors being the same, the multiple benefits of using RFPs make these tools seemingly ideal candidates for use in neurons and, ultimately, the brain. However, for many applications, the practical utility of RFPs still falls short of the preferred GFPs. We present an overview of RFPs and RFP-based biosensors, with an emphasis on their reported applications in neuroscience.

Keywords: calcium ion; fluorescence imaging; genetically encoded biosensors; neurotransmitters; red fluorescent protein; voltage.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Conversion of the wild-type tetrameric red fluorescent protein (RFP) DsRed to an engineered monomeric RFP. (a) Cartoon representation of the structure of wild-type tetrameric RFP DsRed. (b) Disruption of the first A–B interface produces an A–C dimer intermediate and subsequent disruption of the A–C interface produces a monomeric RFP. Interface-disrupting mutations are typically detrimental to the proper folding and chromophore maturation of the intermediate dimer or monomer; therefore, these variants must be rescued by directed evolution. Cartoon structures are based on PDB ID 1G7K.
Fig. 2
Fig. 2
Structure of a representative monomeric RFP, mCherry. The secondary structure is shown in a cartoon representation with the helix colored in yellow, β-strands colored in red, and loops colored in orange. The chromophore is shown in a stick representation with carbon atoms colored in gray, nitrogen atoms colored in blue, and oxygen atoms colored in red (PDB ID 2H5Q).
Fig. 3
Fig. 3
Branched pathway mechanism for red chromophore formation (and dead-end green chromophore formation) in DsRed.
Fig. 4
Fig. 4
Biosensor design based on RFP complementation. Two potentially interacting proteins are fused to the two fragments of a split RFP. Interaction between the two protein partners bring the RFP fragments in close proximity, leading to reconstitution of an intact RFP and a corresponding increase in red fluorescence.
Fig. 5
Fig. 5
Representative Förster resonance energy transfer (FRET)-based biosensors with RFPs. (a) Intermolecular biosensors for protein–protein interaction. Unlike FP complementation-based biosensors, the FRET-based biosensors of protein–protein interactions are reversible. (b) Ion/small molecule biosensors. An intramolecular protein complex is formed, or a conformation changed, upon the binding of a specific ion or small molecule. (c) Protease biosensors where the two FPs are initially linked by a protease substrate sequence.
Fig. 6
Fig. 6
ddRFPs and ddRFP-based biosensors. (a) Fluorescence intensity increase upon the formation of heterodimeric ddRFP pair. (b) ddRFP-based caspase-3 biosensor. (c) ddRFP-based Ca2+ biosensor.
Fig. 7
Fig. 7
Single FP-based biosensors. (a) Single FP-based pH biosensor based on intrinsic sensitivity. (b) Single FP-based Ca2+ biosensor with an extrinsic Ca2+ binding domain.
Fig. 8
Fig. 8
Schematic presentation of FP circular permutation at both the DNA and protein levels.
See this image and copyright information in PMC

References

    1. Shimomura O., Johnson F. H., Saiga Y., “Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea,” J. Cell. Comp. Physiol. 59(3), 223–239 (1962).JCCPAY10.1002/(ISSN)1553-0809 - DOI - PubMed
    1. Shimomura O., “Structure of the chromophore of Aequorea green fluorescent protein,” FEBS Lett. 104(2), 220–222 (1979).FEBLAL10.1016/0014-5793(79)80818-2 - DOI
    1. Prasher D. C., et al. , “Primary structure of the Aequorea victoria green-fluorescent protein,” Gene 111(2), 229–233 (1992).GENED610.1016/0378-1119(92)90691-H - DOI - PubMed
    1. Chalfie M., et al. , “Green fluorescent protein as a marker for gene-expression,” Science 263(5148), 802–805 (1994).SCIEAS10.1126/science.8303295 - DOI - PubMed
    1. Heim R., Prasher D. C., Tsien R. Y., “Wavelength mutations and posttranslational autoxidation of green fluorescent protein,” Proc. Natl. Acad. Sci. U. S. A. 91(26), 12501–12504 (1994).PNASA610.1073/pnas.91.26.12501 - DOI - PMC - PubMed