publication . Article . 2016

Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

Bryce T. Bajar; Emily S. Wang; Amy J. Lam; Bongjae B. Kim; Conor L. Jacobs; Elizabeth S. Howe; Michael W. Davidson; Michael Z. Lin; Jun Chu;
Open Access English
  • Published: 16 Feb 2016 Journal: Scientific Reports, volume 6 (issn: 2045-2322, eissn: 2045-2322, Copyright policy)
  • Publisher: Nature Publishing Group
Abstract
Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostabil...
Subjects
free text keywords: Article
Funded by
NIH| TRAINING & OUTREACH CORE
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1P50GM107615-01
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Protein voltage sensors: kilohertz imaging of neural dynamics in behaving animals
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 3U01NS090600-03S1
  • Funding stream: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
37 references, page 1 of 3

Lam A. J.et al.Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).22961245 [OpenAIRE] [PubMed]

Miranda J. G.et al.New alternately colored FRET sensors for simultaneous monitoring of Zn 2+ in multiple cellular locations. PLoS One 7, e49371 (2012).23173058 [OpenAIRE] [PubMed]

Ni Q., Titov D. V. & Zhang J. Analyzing protein kinase dynamics i n living cells with FRET reporters. Methods 40, 279–286 (2006).16908183 [PubMed]

Takao K.et al.Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons. J Neurosci 25, 3107–3112 (2005).15788767 [OpenAIRE] [PubMed]

Tsutsui H., Karasawa S., Okamura Y. & Miyawaki A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat Methods 5, 683–685 (2008).18622396 [PubMed]

Zhang J., Ma Y., Taylor S. S. & Tsien R. Y. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci USA 98, 14997–15002 (2001).11752448 [OpenAIRE] [PubMed]

Dixit R. & Cyr R. Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J 36, 280–290 (2003).14535891 [PubMed]

Malkani N. & Schmid J. A. Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation. PLoS One 6, e18586 (2011).21490932 [OpenAIRE] [PubMed]

Raarup M. K.et al.Enhanced yellow fluorescent protein photoconversion to a cyan fluorescent protein-like species is sensitive to thermal and diffusion conditions. J Biomed Opt 14, 034039 (2009).19566331 [OpenAIRE] [PubMed]

Reiff D. F.et al.In vivo performance of genetically encoded indicators of neural activity in flies. J Neurosci 25, 4766–4778 (2005).15888652 [OpenAIRE] [PubMed]

Shaner N. C.et al.Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5, 545–551 (2008).18454154 [OpenAIRE] [PubMed]

Sinnecker D., Voigt P., Hellwig N. & Schaefer M. Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44, 7085–7094 (2005).15865453 [PubMed]

Hockberger P. E.et al.Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc Natl Acad Sci USA 96, 6255–6260 (1999).10339574 [OpenAIRE] [PubMed]

George Abraham B.et al.Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements. PLoS One 10, e0134436 (2015).26237400 [OpenAIRE] [PubMed]

Helmchen F. & Denk W. Deep tissue two-photon microscopy. Nat Methods 2, 932–940 (2005).16299478 [OpenAIRE] [PubMed]

37 references, page 1 of 3
Abstract
Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostabil...
Subjects
free text keywords: Article
Funded by
NIH| TRAINING & OUTREACH CORE
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1P50GM107615-01
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Protein voltage sensors: kilohertz imaging of neural dynamics in behaving animals
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 3U01NS090600-03S1
  • Funding stream: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
37 references, page 1 of 3

Lam A. J.et al.Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).22961245 [OpenAIRE] [PubMed]

Miranda J. G.et al.New alternately colored FRET sensors for simultaneous monitoring of Zn 2+ in multiple cellular locations. PLoS One 7, e49371 (2012).23173058 [OpenAIRE] [PubMed]

Ni Q., Titov D. V. & Zhang J. Analyzing protein kinase dynamics i n living cells with FRET reporters. Methods 40, 279–286 (2006).16908183 [PubMed]

Takao K.et al.Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons. J Neurosci 25, 3107–3112 (2005).15788767 [OpenAIRE] [PubMed]

Tsutsui H., Karasawa S., Okamura Y. & Miyawaki A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat Methods 5, 683–685 (2008).18622396 [PubMed]

Zhang J., Ma Y., Taylor S. S. & Tsien R. Y. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci USA 98, 14997–15002 (2001).11752448 [OpenAIRE] [PubMed]

Dixit R. & Cyr R. Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J 36, 280–290 (2003).14535891 [PubMed]

Malkani N. & Schmid J. A. Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation. PLoS One 6, e18586 (2011).21490932 [OpenAIRE] [PubMed]

Raarup M. K.et al.Enhanced yellow fluorescent protein photoconversion to a cyan fluorescent protein-like species is sensitive to thermal and diffusion conditions. J Biomed Opt 14, 034039 (2009).19566331 [OpenAIRE] [PubMed]

Reiff D. F.et al.In vivo performance of genetically encoded indicators of neural activity in flies. J Neurosci 25, 4766–4778 (2005).15888652 [OpenAIRE] [PubMed]

Shaner N. C.et al.Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5, 545–551 (2008).18454154 [OpenAIRE] [PubMed]

Sinnecker D., Voigt P., Hellwig N. & Schaefer M. Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44, 7085–7094 (2005).15865453 [PubMed]

Hockberger P. E.et al.Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc Natl Acad Sci USA 96, 6255–6260 (1999).10339574 [OpenAIRE] [PubMed]

George Abraham B.et al.Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements. PLoS One 10, e0134436 (2015).26237400 [OpenAIRE] [PubMed]

Helmchen F. & Denk W. Deep tissue two-photon microscopy. Nat Methods 2, 932–940 (2005).16299478 [OpenAIRE] [PubMed]

37 references, page 1 of 3
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publication . Article . 2016

Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

Bryce T. Bajar; Emily S. Wang; Amy J. Lam; Bongjae B. Kim; Conor L. Jacobs; Elizabeth S. Howe; Michael W. Davidson; Michael Z. Lin; Jun Chu;