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An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging.
J Am Chem Soc 2012; 134(18):7913-23JA

Abstract

Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Förster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.

Authors+Show Affiliations

Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22486524

Citation

Shcherbakova, Daria M., et al. "An Orange Fluorescent Protein With a Large Stokes Shift for Single-excitation Multicolor FCCS and FRET Imaging." Journal of the American Chemical Society, vol. 134, no. 18, 2012, pp. 7913-23.
Shcherbakova DM, Hink MA, Joosen L, et al. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J Am Chem Soc. 2012;134(18):7913-23.
Shcherbakova, D. M., Hink, M. A., Joosen, L., Gadella, T. W., & Verkhusha, V. V. (2012). An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. Journal of the American Chemical Society, 134(18), pp. 7913-23. doi:10.1021/ja3018972.
Shcherbakova DM, et al. An Orange Fluorescent Protein With a Large Stokes Shift for Single-excitation Multicolor FCCS and FRET Imaging. J Am Chem Soc. 2012 May 9;134(18):7913-23. PubMed PMID: 22486524.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. AU - Shcherbakova,Daria M, AU - Hink,Mark A, AU - Joosen,Linda, AU - Gadella,Theodorus W J, AU - Verkhusha,Vladislav V, Y1 - 2012/04/24/ PY - 2012/4/11/entrez PY - 2012/4/11/pubmed PY - 2012/8/28/medline SP - 7913 EP - 23 JF - Journal of the American Chemical Society JO - J. Am. Chem. Soc. VL - 134 IS - 18 N2 - Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Förster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype. SN - 1520-5126 UR - https://www.unboundmedicine.com/medline/citation/22486524/An_orange_fluorescent_protein_with_a_large_Stokes_shift_for_single_excitation_multicolor_FCCS_and_FRET_imaging_ L2 - https://dx.doi.org/10.1021/ja3018972 DB - PRIME DP - Unbound Medicine ER -